Methods for treating myelodysplastic syndromes and sideroblastic anemias

ABSTRACT

In certain aspects, the present disclosure provides compositions and methods for increasing red blood cell and/or hemoglobin levels in vertebrates, including rodents and primates, and particularly in humans. In some embodiments, the compositions of the disclosure may be used to treat or prevent sideroblastic anemias and myelodysplastic syndromes or one or more complications associated sideroblastic anemias and myelodysplastic syndromes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisionalapplication Ser. No. 62/086,977, filed Dec. 3, 2014; U.S. provisionalapplication Ser. No. 62/088,087, filed Dec. 5, 2014; and U.S.provisional application Ser. No. 62/155,395, filed Apr. 30, 2015. Thedisclosures of each of the foregoing applications are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present disclosure relates to treatments for dysregulated productionof blood cellular components, including red blood cells, neutrophils,and platelets. Hematopoiesis is the formation of cellular components ofthe blood from self-renewing hematopoietic stem cells located mainly inthe bone marrow, spleen, or lymph nodes during postnatal life. Bloodcells can be classified as belonging to the lymphocytic lineage,myelocytic lineage, or erythroid lineage. By a process known aslymphopoiesis, common lymphoid progenitor cells give rise to T-cells,B-cells, natural killer cells, and dendritic cells. By a process termedmyelopoiesis, common myeloid progenitor cells give rise to macrophages,granulocytes (basophils, neutrophils, eosinophils, and mast cells), andthrombocytes (platelets). Finally, by a process known as erythropoiesis,erythroid progenitor cells give rise to red blood cells (RBC,erythrocytes).

Postnatal erythropoiesis occurs primarily in the bone marrow and in thered pulp of the spleen. The coordinated action of various signalingpathways controls the balance of cell proliferation, differentiation,survival, and death. Under normal conditions, red blood cells areproduced at a rate that maintains a constant red cell mass in the body,and production may increase or decrease in response to various stimuli,including increased or decreased oxygen tension or tissue demand. Theprocess of erythropoiesis begins with the formation of lineage-committedprecursor cells and proceeds through a series of distinct precursor celltypes. The final stages of erythropoiesis occur as reticulocytes arereleased into the bloodstream and lose their mitochondria and ribosomeswhile assuming the morphology of mature red blood cell. An elevatedlevel of reticulocytes, or an elevated reticulocyte:erythrocyte ratio,in the blood is indicative of increased red blood cell production rates.The mature red blood cell (RBC) is responsible for oxygen transport inthe circulatory systems of vertebrates. Red blood cells contain highconcentrations of hemoglobin, a protein that binds to oxygen in thelungs at relatively high partial pressure of oxygen (pO2) and deliversoxygen to areas of the body with relatively low pO2.

Erythropoietin (EPO) is widely recognized as a significant positiveregulator of postnatal erythropoiesis in vertebrates. EPO regulates thecompensatory erythropoietic response to reduced tissue oxygen tension(hypoxia) and low red blood cell levels or low hemoglobin levels. Inhumans, elevated EPO levels promote red blood cell formation bystimulating the generation of erythroid progenitors in the bone marrowand spleen. In the mouse, EPO enhances erythropoiesis primarily in thespleen.

Effects of EPO are mediated by a cell-surface receptor belonging to thecytokine receptor superfamily. The human EPO receptor gene encodes a 483amino acid transmembrane protein; however, the active EPO receptor isthought to exist as a multimeric complex even in the absence of ligand(see, e.g., U.S. Pat. No. 6,319,499). The cloned full-length EPOreceptor expressed in mammalian cells binds EPO with an affinity similarto that of the native receptor on erythroid progenitor cells. Binding ofEPO to its receptor causes a conformational change resulting in receptoractivation and biological effects including increased proliferation ofimmature erythroblasts, increased differentiation of immatureerythroblasts, and decreased apoptosis in erythroid progenitor cells[see, e.g., Liboi et al. (1993) Proc Natl Acad Sci USA 90:11351-11355;Koury et al. (1990) Science 248:378-381].

Various forms of recombinant EPO are used by physicians to increase redblood cell levels in a variety of clinical settings, particularly in thetreatment of anemia. Anemia is a broadly-defined condition characterizedby lower than normal levels of hemoglobin or red blood cells in theblood. In some instances, anemia is caused by a primary disorder in theproduction or survival of red blood cells (e.g., myelodysplasticsyndromes). More commonly, anemia is secondary to diseases of othersystems [see, e.g., Weatherall & Provan (2000) Lancet 355, 1169-1175].Anemia may result from a reduced rate of production or increased rate ofdestruction of red blood cells or by loss of red blood cells due tobleeding. Anemia may result from a variety of disorders that include,for example, acute or chronic renal failure or end stage renal disease,chemotherapy treatment, a myelodysplastic syndrome, rheumatoidarthritis, and bone marrow transplantation.

Treatment with EPO typically causes a rise in hemoglobin by about 1-3g/dL in healthy humans over a period of weeks. When administered toanemic individuals, this treatment regimen often provides substantialincreases in hemoglobin and red blood cell levels and leads toimprovements in quality of life and prolonged survival. However, EPO isnot uniformly effective, and many individuals are refractory to evenhigh doses [see, e.g., Horl et al. (2000) Nephrol Dial Transplant 15,43-50]. For example, over 50% of patients with cancer have an inadequateresponse to EPO, and approximately 10% with end-stage renal disease arehyporesponsive to EPO [see, e.g., Glaspy et al. (1997) J Clin Oncol 15,1218-1234; Demetri et al. (1998) J Clin Oncol 16, 3412-3425]. Althoughthe molecular mechanisms of resistance to EPO are as yet unclear,several factors, including inflammation, iron and vitamin deficiency,inadequate dialysis, aluminum toxicity, and hyperparathyroidism maypredict a poor therapeutic response. In addition, recent evidencesuggests that higher doses of EPO may be associated with an increasedrisk of cardiovascular morbidity, tumor growth, and mortality in somepatient populations [see, e.g., Krapf et al. (2009) Clin J Am SocNephrol 4:470-480; Glaspy (2009) Annu Rev Med 60:181-192]. Therefore, ithas been recommended that EPO-based therapeutic compounds (e.g.,erythropoietin-stimulating agents, ESAs) be administered at the lowestdose that allows a patient to avoid red blood cell transfusions [see,e.g., Jelkmann et al. (2008) Crit Rev Oncol. Hematol 67:39-61].

Sideroblastic anemia, which occurs in both inherited and acquired forms,is characterized by the presence of “ring sideroblasts” in bone marrow.These distinctive red blood cell precursors (erythroblasts) can beidentified by the presence of perinuclear siderotic granules, which arerevealed by histologic staining with Prussian blue and are indicative ofpathologic iron deposits in mitochondria [see, e.g., Mufti et al. (2008)Haematologica 93:1712-1717; Bottomley et al. (2014) Hematol Oncol Clin NAm 28:653-670]. Acquired sideroblastic anemia occurs most frequently inthe context of myelodysplastic syndromes (MDS), a heterogeneous group ofhematopoietic stem-cell disorders estimated to affect between 30,000 and40,000 patients per year in the United States [Bejar et al. (2014) Blood124:2793-2803]. These disorders are characterized by ineffectivehematopoiesis, abnormal “dysplastic” cell morphology, and the potentialfor clonal evolution to acute myeloid leukemia. As discussed below,recent advances in the genetic basis of MDS have the potential togreatly improve its diagnosis and treatment.

There is high unmet need for effective therapies for MDS, sideroblasticanemia and complications of those disorders. Endogenous EPO levels arecommonly elevated in subsets of patients with MDS, thus suggesting thatEPO has diminished effectiveness in these patients. It has beenestimated that fewer than 10% of patients with MDS respond favorably toEPO [Estey (2003) Curr Opin Hematol 10, 60-67], while a more recentmeta-analysis found that EPO response rates range from 30% to 60%depending on the study [Moyo et al (2008) Ann Hematol 87:527-536].Compared to other MDS patients, those with ring sideroblasts tend to beat substantially lower risk of developing acute myeloid leukemia andwould therefore stand to benefit for an extended period from anti-anemiatherapeutic agents that do not contribute to systemic iron burden andthat instead help to reduce the iron overload frequently present in suchpatients [see, e.g., Temraz et al., 2014, Crit Rev Oncol Hematol91:64-73].

Thus, it is an object of the present disclosure to provide methodstreating patients with MDS and sideroblastic anemias with ActRIIantagonists disclosed herein and, in particular, to guide selection ofMDS patients that are most likely to show therapeutically beneficialincreases in red blood cells, neutrophils, and other blood cells as aresult of treatment.

SUMMARY OF THE INVENTION

In part, the disclosure provides methods of treating MDS andsideroblastic anemias, particularly treating or preventing one or morecomplications or subtypes of MDS, including MDS patients characterizedby the presence of sideroblasts in the bone marrow, with one or moreActRII antagonists.

In part the disclosure provides methods for treating or preventingdisorders or complications of a disorder that is associated with germline or somatic mutations in SF3B1, such as myelodysplastic syndrome(MDS), chronic lymphocytic leukemia (CLL), and acute myeloid leukemia(AML) as well as in breast cancer, pancreatic cancer, gastric cancer,prostate cancer, and uveal melanoma with one or more ActRII antagonists.In certain aspects the disorder may be in a subject that has bone marrowcells that test positive for an SF3B1 mutation, particularlymyelodysplastic syndrome, CLL and AML. Optionally a mutation in theSF3B1 gene is in an exon, intron or 5′ and/or 3′ untranslated region.For example, in some embodiments, a mutation in the SF3B1 gene is inexon 14, 15, and/or 16 of SF31B. Optionally a mutation in SF3B1 causes achange in the amino acid sequence or does not cause a change in theamino acid sequence of the protein encoded by the gene. Optionally amutation in the SF3B1 gene causes a change in the amino acid of theprotein encoded by the gene selected from the following changes: K182E,E491G, R590K, E592K, R625C, R625G, N626D, N626S, H662Y, T663A, K666M,K666Q, K666R, Q670E, G676D, V701I, I1704N, 1704V, G740R, A744P, D781G,A1188V, N619K, N626H, N626Y, R630S, 1704T, G740E, K741N, G742D, D894G,Q903R, R1041H, I1241T, G347V, E622D, Y623C, R625H, R625L, H662D, H662Q,T663I, K666E, K666N, K666T, K700E, E783K, and V701F.

In certain aspects, the disclosure provides methods for treating orpreventing sideroblastic anemia in a human subject, comprisingadministering to a subject in need thereof a polypeptide comprising anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids29-109 of SEQ ID NO: 1, wherein the polypeptide comprises an acidicamino acid [a naturally occurring amino acid (e.g., D or E) or anartificial amino acid] at position 79 with respect to SEQ ID NO: 1,wherein the subject is on a dosing schedule that comprisingadministering from 0.125 to 1.75 mg/kg (e.g., 0.75 to 1.75 mg/kg) of thepolypeptide to the subject. In other aspects, the disclosure providesmethods for treating or preventing sideroblastic anemia in a humansubject, comprising administering to a subject in need thereof apolypeptide comprising an amino acid sequence that is at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 25-125 of SEQ ID NO: 1, wherein the polypeptidecomprises an acidic amino acid [a naturally occurring amino acid (e.g.,D or E) or an artificial amino acid] at position 79 with respect to SEQID NO: 1, wherein the subject is on a dosing schedule that comprisingadministering from 0.125 to 1.75 mg/kg (e.g., 0.75 to 1.75 mg/kg) of thepolypeptide to the subject. In even other aspects, the disclosureprovides methods for treating or preventing sideroblastic anemia in ahuman subject, comprising administering to a subject in need thereof apolypeptide comprising, consisting essentially of, or consisting of anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acidsequence of SEQ ID NO: 44, wherein the polypeptide comprises an acidicamino acid [a naturally occurring amino acid (e.g., D or E) or anartificial amino acid] at position 79 with respect to SEQ ID NO: 1,wherein the subject is on a dosing schedule that comprisingadministering from 0.125 to 1.75 mg/kg (e.g., 0.75 to 1.75 mg/kg) of thepolypeptide to the subject. In certain aspects, the disclosure providesmethods for treating or preventing one or more complications ofsideroblastic anemia in a human subject, comprising administering to asubject in need thereof a polypeptide comprising an amino acid sequencethat is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ ID NO: 1,wherein the polypeptide comprises an acidic amino acid [a naturallyoccurring amino acid (e.g., D or E) or an artificial amino acid] atposition 79 with respect to SEQ ID NO: 1, wherein the subject is on adosing schedule that comprising administering from 0.125 to 1.75 mg/kg(e.g., 0.75 to 1.75 mg/kg) of the polypeptide to the subject. In otheraspects, the disclosure provides methods for treating or preventingand/or one or more complications of sideroblastic anemia in a humansubject, comprising administering to a subject in need thereof apolypeptide comprising an amino acid sequence that is at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to amino acids 25-125 of SEQ ID NO: 1, wherein the polypeptidecomprises an acidic amino acid [a naturally occurring amino acid (e.g.,D or E) or an artificial amino acid] at position 79 with respect to SEQID NO: 1, wherein the subject is on a dosing schedule that comprisingadministering from 0.125 to 1.75 mg/kg (e.g., 0.75 to 1.75 mg/kg) of thepolypeptide to the subject. In even other aspects, the disclosureprovides methods for treating or preventing and/or one or morecomplications of sideroblastic anemia in a human subject, comprisingadministering to a subject in need thereof a polypeptide comprising,consisting essentially of, or consisting of an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 44,wherein the polypeptide comprises an acidic amino acid [a naturallyoccurring amino acid (e.g., D or E) or an artificial amino acid] atposition 79 with respect to SEQ ID NO: 1, wherein the subject is on adosing schedule that comprising administering from 0.125 to 1.75 mg/kg(e.g., 0.75 to 1.75 mg/kg) of the polypeptide to the subject. In certainpreferred embodiments, polypeptides to be used in accordance with themethods described herein are dimers (e.g., a homodimer comprising twopolypeptides corresponding to the amino acid sequence of SEQ ID NO: 44associated by covalent or non-covalent interactions). Optionally thepolypeptide may bind to one or more ligand of the TGFβ superfamily. Forexample, in some embodiments, polypeptides described herein (e.g.,ActRIIA and ActRIIB polypeptides as well as variant thereof such as GDFtraps) may bind to GDF11. In other embodiments, polypeptides describedherein (e.g., ActRIIA and ActRIIB polypeptides as well as variantthereof such as GDF traps) may bind to GDF8. In still other embodiments,polypeptides described herein (e.g., ActRIIA and ActRIIB polypeptides aswell as variant thereof such as GDF traps) may bind to GDF11 and GDF8.Optionally the polypeptide may comprises one or more amino acidmodifications selected from: a glycosylated amino acid, a PEGylatedamino acid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, and an amino acid conjugated to a lipid moiety.In certain preferred embodiments the polypeptide is glycosylated and hasa mammalian glycosylation pattern. Optionally the polypeptide has aglycosylation pattern obtainable from a Chinese hamster ovary cell line.Optionally the methods comprises subcutaneously administered thepolypeptide to the subject. Optionally the dosing schedule furthercomprises administering the polypeptide to the patient twice every week,once every week, once every 3 weeks, once every 4 weeks, once every 5weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, onceevery 9 weeks, once every 10 weeks, once every 12 weeks, once every 14weeks, once every 16 weeks, once every 18 weeks, once every 20 weeks,once every 24 weeks, once every 26 weeks, once every 28 weeks, onceevery 30 weeks, once every 32 weeks, once every 34 weeks, or once every36 weeks In certain preferred embodiments the dosing schedule furthercomprises administering the polypeptide to the patient once every threeweeks. Optionally the subject has undesirably high levels of endogenousEPO. Optionally the subject has previously been treated with one or moreEPO receptor agonists. Optionally the subject has an inadequate responseto the EPO receptor agonist. Optionally the subject is no longerresponsive to the EPO receptor agonist. Optionally the EPO receptoragonist is EPO. Optionally the treatment increases red blood celllevels. Optionally the treatment increases hemoglobin levels. Optionallywherein the treatment results in a hemoglobin increase of ≥1.5 g/dL for≥two weeks. Optionally the treatment results in a hemoglobin increase of≥1.5 g/dL for ≥eight weeks. Optionally the subject has been administeredone or more blood cell transfusions prior to the start of treatment.Optionally wherein the subject is a low transfusion burden subject.Optionally the subject is a high transfusion burden subject. Optionallythe treatment decreases blood cell transfusion burden. Optionally thetreatment decreases blood cell transfusion by ≥50% for at least fourweeks relative to the equal time prior to start of treatment. Optionallythe treatment decreases blood cell transfusion by ≥50% for at leasteight weeks relative to the equal time prior to start of treatment.Optionally the patient has myelodysplastic syndrome. Optionally thepatient has an International Prognostic Scoring System (IPSS) or IPSS-Rscore of low or intermediate. Optionally the sideroblastic anemiasubject has at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% ring blasts as a percentage of bonemarrow erythroid precursors in his or her bone marrow. Optionally thetreatment increases neutrophil levels. Optionally the subject has bonemarrow cells that test positive for one or more mutations in SF3B1.Optionally the subject has bone marrow cells that test positive for oneor more mutations in DNMT3A. Optionally the subject has bone marrowcells that test positive for one or more mutations in TET2. Optionallythe treatment decreases iron overload. In some embodiments, thetreatment decrease tissue iron overload (e.g., iron overload in thekidney, liver, and/or spleen). In some embodiments, the treatmentdecrease serum iron overload.

In part the disclosure provides methods for treating or preventingdisorders or complications of a disorder that is associated with germline or somatic mutations in DNMT3A, such as myelodysplastic syndrome(MDS), chronic lymphocytic leukemia (CLL), and acute myeloid leukemia(AML) with one or more ActRII antagonists. In certain aspects thedisorder may be in a subject that has bone marrow cells that testpositive for a DNMT3A mutation, particularly myelodysplastic syndrome,CLL and AML. Optionally a mutation in the DNMT3A gene is in an exon,intron and/or 5′ or 3′ untranslated region. For example, in someembodiments, a mutation in the DNMT3A gene is in exon 10, 18, and/or 22of DNMT3A. Optionally a mutation in DNMT3A causes a change in the aminoacid sequence or does not cause a change in the amino acid sequence ofthe protein encoded by the gene. Optionally a mutation in the DNMT3Agene causes a change in the amino acid of the protein encoded by thegene selected from the following changes: R882C, R882H, P904L, andP905P. Optionally a mutation in the DNMT3A gene introduces a prematurestop codon. For example, in some embodiments, a mutation in the DNMT3Agene that introduces a premature stop codon is selected from thefollowing positions: Y436X and W893X.

In part the disclosure provides methods for treating or preventingdisorders or complications of a disorder that is associated with germline or somatic mutations in TET2, such as myelodysplastic syndrome(MDS), chronic lymphocytic leukemia (CLL), and acute myeloid leukemia(AML) with one or more ActRII antagonists. In certain aspects thedisorder may be in a subject that has bone marrow cells that testpositive for a TET2 mutation, particularly myelodysplastic syndrome, CLLand AML. Optionally a mutation in the TET2 gene is in an exon, intronand/or 5′ or 3′ untranslated region. For example, in some embodiments, amutation in the TET2 gene is in exon 1, exon 4, exon 5, exon 6, exon 7,exon 8, and/or exon 9 of TET2. Optionally a mutation in TET2 causes achange in the amino acid sequence or does not cause a change in theamino acid sequence of the protein encoded by the gene. Optionally amutation in the TET2 gene causes a change in the amino acid of theprotein encoded by the gene selected from the following changes: E47Q,Q1274R, W1291R, G1370R, N1387S, and Y1724H. Optionally a mutation in theTET2 gene introduces a premature stop codon. For example, in someembodiments, a mutation in the TET2 gene that introduces a prematurestop codon is selected from the following positions: R550X, Q1009X,Y1337X, R1404X, R1516X, and Q1652X.

In certain aspects, the disclosure provides methods for treating orpreventing a bone marrow disorder in a subject, comprising administeringto a subject in need thereof an effective amount of an ActRIIantagonist, wherein the subject has bone marrow cells that test positivefor one or more mutations in a gene selected from the group consistingof: SF3B1, DNMT3A, and TET2. In some embodiments, the subject testspositive for one or more SF3B1 mutations. Optionally one or more of theSF3B1 mutations are in a SF3B1 exon. Optionally one or more of the SF3B1mutations are in a SF3B1 intron. Optionally one or more of the SF3B1mutations are in a SF3B1 5′ and/or 3′ region. Optionally one or more ofthe SF3B1 mutations causes a deletion, addition, and/or substitution ofan amino acid in the protein encoded by the mutated SF3B1 gene.Optionally one or more SF3B1 mutations causes a substitution of one ormore amino acid selected from the group consisting of: K182E, E491G,R590K, E592K, R625C, R625G, N626D, N626S, H662Y, T663A, K666M, K666Q,K666R, Q670E, G676D, V701I, I704N, 1704V, G740R, A744P, D781G, A1188V,N619K, N626H, N626Y, R630S, 1704T, G740E, K741N, G742D, D894G, Q903R,R1041H, I1241T, G347V, E622D, Y623C, R625H, R625L, H662D, H662Q, T663I,K666E, K666N, K666T, K700E, V701F, and E783K. Optionally one or moreSF3B1 mutations are in a SF3B1 exon selected from the group consistingof: exon 14, exon 15 and exon 16. In some embodiment, the subject testspositive for one or more DNMT3A mutations. Optionally one or more of theDNMT3A mutations are in a DNMT3A exon. Optionally one or more of theDNMT3A mutations are in a DNMT3A intron. Optionally one or more of theDNMT3A mutations are in a DNMT3A 5′ and/or 3′ region. Optionally one ormore of the DNMT3A mutations causes a deletion, addition, and/orsubstitution of an amino acid in the protein encoded by the mutatedDNMT3A gene. Optionally one or more DNMT3A mutations causes asubstitution of one or more amino acid selected from the groupconsisting of: R882C, R882H, P904L, and P905P. Optionally the one ormore DNMT3A mutations are in a DNMT3A exon selected from the groupconsisting of: exon 10, exon 18 and exon 22. Optionally one or moreDNMT3A mutations introduces a premature stop codon. Optionally one ormore DNMT3A mutations is selected from the group consisting of Y436X andW893X. In some embodiments, subject tests positive for one or more TET2mutations. Optionally one or more of the TET2 mutations are in a TET2exon. Optionally one or more of the TET2 mutations are in a TET2 intron.Optionally one or more of the TET2 mutations are in a TET2 5′ and/or 3′region. Optionally one or more of the TET2 mutations causes a deletion,addition, and/or substitution of an amino acid in the protein encoded bythe mutated TET2 gene. Optionally one or more TET2 mutations causes asubstitution of one or more amino acid selected from the groupconsisting of: E47Q, Q1274R, W1291R, G1370R, G1370E, N1387S, and Y1724H.Optionally one or more TET2 mutations are in a TET2 exon selected fromthe group consisting of: exon 1, exon 4, exon 5, exon 6, exon 7, exon 8,and exon 9. Optionally one or more TET2 mutations introduces a prematurestop codon. Optionally one or more TET2 mutations is selected from thegroup consisting of: R550X, Q1009X, Y1337X, R1404X, R1516X, and Q1652X.Optionally the subject has anemia. Optionally the subject hasundesirably high levels of endogenous EPO. Optionally the subject haspreviously been treated with one or more EPO receptor agonists.Optionally the subject has an inadequate response to the EPO receptoragonist. Optionally the subject is no longer responsive to the EPOreceptor agonist. Optionally the EPO receptor agonist is EPO. Optionallythe treatment delays conversion to leukemia. Optionally the treatmentdelays conversion to acute myeloid leukemia. Optionally the subject is apre-leukemia patient. Optionally the treatment increases red blood celllevels and/or hemoglobin levels in the subject. Optionally the subjecthas been administered one or more blood cell transfusions prior to thestart of the ActRII antagonist treatment. Optionally the treatmentdecreases blood cell transfusion burden. Optionally the treatmentdecreases blood cell transfusion by greater than about 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% for 4 to 8 weeks relative to the equal timeprior to the start of the ActRII antagonist treatment. Optionally thetreatment decreases blood cell transfusion by greater than about 50% for4 to 8 weeks relative to the equal time prior to the start of the ActRIIantagonist treatment. Optionally the treatment decreases iron overload.Optionally the treatment decrease iron content in the liver and/orspleen.

In certain aspects, the disclosure provides methods for treating orpreventing myelodysplastic syndrome (MDS) and/or one or morecomplications of MDS, comprising administering to a subject in needthereof an effective amount of an ActRII antagonist, wherein the subjecthas bone marrow cells that test positive for one or more mutations in agene selected from the group consisting of: SF3B1, DNMT3A, and TET2. Insome embodiments, the subject tests positive for one or more SF3B1mutations. Optionally one or more of the mutations are in a SF3B1 exon.Optionally one or more of the SF3B1 mutations are in a SF3B1 intron.Optionally one or more of the SF3B1 mutations are in a SF3B1 5′ and/or3′ region. Optionally one or more of the SF3B1 mutations causes adeletion, addition, and/or substitution of an amino acid in the proteinencoded by the mutated SF3B1 gene. Optionally one or more SF3B1mutations causes a substitution of one or more amino acid selected fromthe group consisting of: K182E, E491G, R590K, E592K, R625C, R625G,N626D, N626S, H662Y, T663A, K666M, K666Q, K666R, Q670E, G676D, V701I,1704N, 1704V, G740R, A744P, D781G, A1188V, N619K, N626H, N626Y, R630S,1704T, G740E, K741N, G742D, D894G, Q903R, R1041H, I1241T, G347V, E622D,Y623C, R625H, R625L, H662D, H662Q, T663I, K666E, K666N, K666T, K700E,V701F, and E783K. Optionally one or more SF3B1 mutations are in a SF3B1exon selected from the group consisting of: exon 14, exon 14 and exon16. In some embodiments, the subject tests positive for one or moreDNMT3A mutations. Optionally one or more of the DNMT3A mutations are ina DNMT3A exon. Optionally one or more of the DNMT3A mutations are in aDNMT3A intron. Optionally one or more of the DNMT3A mutations are in aDNMT3A 5′ and/or 3′ region. Optionally one or more of the DNMT3Amutations causes a deletion, addition, and/or substitution of an aminoacid in the protein encoded by the mutated DNMT3A gene. Optionally oneor more DNMT3A mutations causes a substitution of one or more amino acidselected from the group consisting of: R882C, R882H, P904L, and P905P.Optionally one or more DNMT3A mutations are in a DNMT3A exon selectedfrom the group consisting of: exon 10, exon 18, and exon 22. Optionallyone or more DNMT3A mutations introduces a premature stop codon.Optionally one or more DNMT3A mutations are selected from the groupconsisting of Y436X and W893X. In some embodiments, the subject testspositive for one or more TET2 mutations. Optionally one or more of theTET2 mutations are in a TET2 exon. Optionally one or more of the TET2mutations are in a TET2 intron. Optionally one or more of the TET2mutations are in a TET2 5′ and/or 3′ region. Optionally one or more ofthe TET2 mutations causes a deletion, addition, and/or substitution ofan amino acid in the protein encoded by the mutated TET2 gene.Optionally one or more TET2 mutations causes a substitution of one ormore amino acid selected from the group consisting of: E47Q, Q1274R,W1291R, G1370R, G1370E, N1387S, and Y1724H. Optionally one or more TET2mutations are in a TET2exon selected from the group consisting of: exon1, exon 4, exon 5, exon 6, exon 7, exon 8, and exon 9. Optionally one ormore TET2 mutations introduces a premature stop codon. Optionally one ormore TET2 mutations is selected from the group consisting of: R550X,Q1009X, Y1337X, R1404X, R1516X, and Q1652X. Optionally the subject hasanemia. Optionally the subject has undesirably high levels of endogenousEPO. Optionally the subject has previously been treated with one or moreEPO receptor agonists. Optionally the subject has an inadequate responseto the EPO receptor agonist. Optionally the subject is no longerresponsive to the EPO receptor agonist. Optionally the EPO receptoragonist is EPO. Optionally the treatment delays conversion to leukemia.Optionally the treatment delays conversion to acute myeloid leukemia.Optionally the treatment increases red blood cell levels and/orhemoglobin levels in the subject. Optionally the subject has beenadministered one or more blood cell transfusions prior to the start ofthe ActRII antagonist treatment. Optionally the treatment decreasesblood cell transfusion burden. Optionally the treatment decreases bloodcell transfusion by greater than about 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100% for 4 to 8 weeks relative to the equal time prior to thestart of the ActRII antagonist treatment. Optionally the treatmentdecreases blood cell transfusion by greater than about 50% for 4 to 8weeks relative to the equal time prior to the start of the ActRIIantagonist treatment. Optionally the treatment decreases iron overload.Optionally the treatment decrease iron content in the liver and/orspleen. Optionally the subject has a subtype of MDS selected from: MDSwith refractory anemia with ring sideroblasts (RARS); MDS withrefractory anemia with ring sideroblasts and thrombocytosis (RARS-T);MDS with refractory cytopenia with unilineage dysplasia (RCUD); MDS withrefractory cytopenia with multilineage dysplasia and ring sideroblasts(RCMD-RS); MDS with a somatic mutation in one or more of SF3B1, SRSF2,DNMT3A, and TET2; MDS without a somatic mutation in ASXL1 or ZRSR2; MDSwith iron overload; and MDS with neutropenia. Optionally the subject hasan International Prognostic Scoring System (IPSS) score selected from:low, intermediate 1, or intermediate 2. Optionally the subject hassideroblasts. Optionally the subject has at least 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%sideroblasts as a percentage of bone marrow erythroid precursors in hisor her bone marrow.

ActRII antagonists of the disclosure include, for example, agents thatcan inhibit ActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor)mediated activation of a signal transduction pathway (e.g., activationof signal transduction via intracellular mediators, such as SMAD 1, 2,3, 5, and/or 8); agents that can inhibit one or more ActRII ligands(e.g., activin A, activin B, activin AB, activin C, activin E, GDF11,GDF8, BMP6, BMP7, Nodal, etc.) from, e.g., binding to and/or activatingan ActRII receptor; agents that inhibit expression (e.g., transcription,translation, cellular secretion, or combinations thereof) of an ActRIIligand and/or an ActRII receptor; and agents that can inhibit one ormore intracellular mediators of the ActRII signaling pathway (e.g.,SMADs 1, 2, 3, 5, and/or 8).

In particular, the disclosure provides methods for using an ActRIIantagonist, or combination of ActRII antagonists, to treat or preventone or more complications of MDS and sideroblastic anemias including,for example, anemia, neutropenia, splenomegaly, blood transfusionrequirement, development of acute myeloid leukemia, iron overload, andcomplications of iron overload, among which are congestive heartfailure, cardiac arrhythmia, myocardial infarction, other forms ofcardiac disease, diabetes mellitus, dyspnea, hepatic disease, andadverse effects of iron chelation therapy and as other examples, toincrease red blood cell levels in a subject in need thereof, treat orprevent an anemia in a subject in need thereof (including, e.g.,reduction of transfusion burden), treat MDS or sideroblastic anemias ina subject in need thereof, and/or treat or prevent one or morecomplications of MDS or sideroblastic anemias (e.g., anemia, bloodtransfusion requirement, neutropenia, iron overload, acute myocardialinfarction, hepatic failure, hepatomegaly, splenomegaly, progression toacute myeloid lymphoma) and or treat or prevent a disorder associatedwith SF3B1, DNMT3A, and/or TET2 mutations in a subject in need thereof.

In particular, the disclosure provides methods for using an ActRIIantagonist, or combination of ActRII antagonists, to treat or preventcomplications in a subtype of MDS, including MDS patients with elevatednumbers of erythroblasts (hypercellularity) in bone marrow; in MDSpatients with more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sideroblasts, as apercentage of erythroid precursors in bone marrow; in MDS patients withrefractory anemia with ring sideroblasts (RARS); in MDS patients withrefractory anemia with ring sideroblasts and thrombocytosis (RARS-T); inMDS patients with refractory cytopenia with unilineage dysplasia (RCUD);in MDS patients with refractory cytopenia with multilineage dysplasiaand ring sideroblasts (RCMD-RS); in MDS patients with a somatic mutationin SF3B1, SRSF2, DNMT3A, TET2, or SETBP1; in MDS patients without asomatic mutation in ASXL1 or ZRSR2; in MDS patients with iron overload;and in MDS patients with neutropenia.

Also in particular, the disclosure provides methods for using an ActRIIantagonist, or combination of ActRII antagonists, to treat or preventcomplications of a sideroblastic anemia, including refractory anemiawith ring sideroblasts (RARS); refractory anemia with ring sideroblastsand thrombocytosis (RARS-T); refractory cytopenia with multilineagedysplasia and ring sideroblasts (RCMD-RS); sideroblastic anemiaassociated with alcoholism; drug-induced sideroblastic anemia;sideroblastic anemia resulting from copper deficiency (zinc toxicity);sideroblastic anemia resulting from hypothermia; X-linked sideroblasticanemia (XLSA); SLC25A38 deficiency; glutaredoxin 5 deficiency;erythropoietic protoporphyria; X-linked sideroblastic anemia with ataxia(XLSA/A); sideroblastic anemia with B-cell immunodeficiency, fevers, anddevelopmental delay (SIFD); Pearson marrow-pancreas syndrome; myopathy,lactic acidosis, and sideroblastic anemia (MLASA); thiamine-responsivemegaloblastic anemia (TRIVIA); and syndromic/nonsyndromic sideroblasticanemia of unknown cause.

In certain embodiments, preferred ActRII antagonists to be used inaccordance with the methods disclosed herein are agents that bind toand/or inhibit GDF11 and/or GDF8 (e.g., an agent that inhibits GDF11-and/or GDF8-mediated activation of ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling). Such agents are referred tocollectively as GDF-ActRII antagonists. Optionally, such GDF-ActRIIantagonists may further inhibit one or more of activin A, activin B,activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, and Nodal.Therefore, in some embodiments, the disclosure provides methods of usingone or more ActRII antagonists, including, for example, soluble ActRIIApolypeptides, soluble ActRIIB polypeptides, GDF trap polypeptides,anti-ActRIIA antibodies, anti-ActRIIB antibodies, anti-ActRII ligandantibodies (e.g, anti-GDF11 antibodies, anti-GDF8 antibodies,anti-activin A antibodies, anti-activin B antibodies, anti-activin ABantibodies, anti-activin C antibodies, anti-activin E antibodies,anti-BMP6 antibodies, anti-BMP7 antibodies, and anti-Nodal antibodies),small-molecule inhibitors of ActRIIA, small-molecule inhibitors ofActRIIB, small-molecule inhibitors of one or more ActRII ligands (e.g.,activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8,BMP6, BMP7, Nodal, etc.), inhibitor nucleotides (nucleotide-basedinhibitors) of ActRIIA, inhibitor nucleotides of ActRIIB, inhibitornucleotides of one or more ActRII ligands (e.g., activin A, activin B,activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.),or combinations thereof, to increase red blood cell levels and/orhemoglobin levels in a subject in need thereof, treat or prevent ananemia in a subject in need thereof, treat sideroblastic anemia or MDSin a subject in need thereof, or treat one or more complications ofsideroblastic anemia or MDS in a subject in need thereof and as otherexamples, to increase red blood cell levels in a subject in needthereof, treat or prevent an anemia in a subject in need thereof(including, e.g., reduction of transfusion burden), treat MDS orsideroblastic anemias in a subject in need thereof, and/or treat orprevent one or more complications of MDS or sideroblastic anemias (e.g.,anemia, blood transfusion requirement, neutropenia, iron overload, acutemyocardial infarction, hepatic failure, hepatomegaly, splenomegaly,progression to acute myeloid lymphoma) and or treat or prevent adisorder associated with SF3B1, DNMT3A, and/or TET2 mutations in asubject in need thereof. In certain embodiments, ActRII antagonists tobe used in accordance with the methods disclosed herein do notsubstantially bind to and/or inhibit activin A (e.g., activin A-mediatedactivation of ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 signaling).

In part, the present disclosure demonstrates that an ActRII antagonistcomprising a variant, extracellular (soluble) ActRIIB domain that bindsto and inhibits GDF11 activity (e.g., GDF11-mediated ActRIIA and/orActRIIB signaling transduction, such as SMAD 2/3 signaling) may be usedto increase red blood cell levels in vivo, treat anemia resulting fromvarious conditions/disorders, and treat sideroblastic anemia or MDS.Therefore, in certain embodiments, preferred ActRII antagonists to beused in accordance with the methods disclosed herein (e.g., methods ofincrease red blood cell levels in a subject in need thereof, treat orprevent an anemia in a subject in need thereof (including, e.g.,reduction of transfusion burden), treat MDS or sideroblastic anemias ina subject in need thereof, and/or treat or prevent one or morecomplications of MDS or sideroblastic anemias (e.g., anemia, bloodtransfusion requirement, neutropenia, iron overload, acute myocardialinfarction, hepatic failure, hepatomegaly, splenomegaly, progression toacute myeloid lymphoma) and or treat or prevent a disorder associatedwith SF3B1 mutations in a subject in need thereof, etc.) are solubleActRII polypeptides (e.g. soluble ActRIIA or ActRIIB polypeptides) thatbind to and/or inhibit GDF11 (e.g., GDF11-mediated activation of ActRIIAand/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).While soluble ActRIIA and soluble ActRIIB ActRII antagonists may affectred blood cell formation and/or morphology through a mechanism otherthan GDF11 antagonism, the disclosure nonetheless demonstrates thatdesirable therapeutic agents, with respect to the methods disclosedherein, may be selected on the basis of GDF11 antagonism or ActRIIantagonism or both. Optionally, such soluble ActRII polypeptideantagonist may further bind to and/or inhibit GDF8 (e.g. inhibitGDF8-mediated activation of ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling). In some embodiments, solubleActRIIA and ActRIIB polypeptides of the disclosure that bind to and/orinhibit GDF11 and/or GDF8 may further bind to and/or inhibit one or moreadditional ActRII ligands selected from: activin A, activin B, activinAB, activin C, activin E, BMP6, BMP7, and Nodal.

In certain aspects, the present disclosure provides GDF traps that arevariant ActRII polypeptides (e.g., ActRIIA and ActRIIB polypeptides),including ActRII polypeptides having amino- and carboxy-terminaltruncations and/or other sequence alterations (one or more amino acidsubstitutions, additions, deletions, or combinations thereof).Optionally, GDF traps of the invention may be designed to preferentiallyantagonize one or more ligands of ActRII receptors, such as GDF8 (alsocalled myostatin), GDF11, Nodal, BMP6, and BMP7 (also called OP-1). Asdisclosed herein, examples of GDF traps include a set of variantsderived from ActRIIB that have greatly diminished affinity for activin,particularly activin A. These variants exhibit desirable effects on redblood cells while reducing effects on other tissues. Examples of suchvariants include those having an acidic amino acid [e.g., aspartic acid(D) or glutamic acid (E)] at the position corresponding to position 79of SEQ ID NO:1. In certain embodiments, preferred GDF traps to be usedin accordance with the methods disclosed herein (e.g., methods toincrease red blood cell levels in a subject in need thereof, treat orprevent an anemia in a subject in need thereof (including, e.g.,reduction of transfusion burden), treat MDS or sideroblastic anemias ina subject in need thereof, and/or treat or prevent one or morecomplications of MDS or sideroblastic anemias (e.g., anemia, bloodtransfusion requirement, neutropenia, iron overload, acute myocardialinfarction, hepatic failure, hepatomegaly, splenomegaly, progression toacute myeloid lymphoma) and or treat or prevent a disorder associatedwith SF3B1, DNMT3A, and/or TET2 mutations in a subject in need thereof,etc.) bind to and/or inhibit GDF11. Optionally, such GDF traps mayfurther bind to and/or inhibit GDF8. In some embodiments, GDF traps thatbind to and/or inhibit GDF11 and/or GDF8 may further bind to and/orinhibit one or more additional ActRII ligands (e.g., activin B, activinE, BMP6, BMP7, and Nodal). In some embodiments, GDF traps to be used inaccordance with the methods disclosed herein to not substantially bindto and/or inhibit activin A (e.g., activin A-mediated activation ofActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3signaling). In certain embodiments, a GDF trap polypeptide comprises anamino acid sequence that comprises, consists of, or consists essentiallyof, the amino acid sequence of SEQ ID NOs: 36, 37, 41, 44, 45, 50 or 51,and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to any of the foregoing. In other embodiments, a GDF trappolypeptide comprises an amino acid sequence that comprises, consistsof, or consists essentially of the amino acid sequence of SEQ ID NOs: 2,3, 4, 5, 6, 10, 11, 22, 26, 28, 29, 31, or 49, and polypeptides that areat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any ofthe foregoing. In still other embodiments, a GDF trap polypeptidecomprises an amino acid sequence that comprises of the amino acidsequence of SEQ ID NOs: 2, 3, 4, 5, 6, 29, 31, or 49, and polypeptidesthat are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toany of the foregoing, wherein the position corresponding to 79 in SEQ IDNO: 1, 4, or 50 is an acidic amino acid. A GDF trap may include afunctional fragment of a natural ActRII polypeptide, such as onecomprising at least 10, 20, or 30 amino acids of a sequence selectedfrom SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9, 10, 11, or 49 or a sequence of SEQID NO: 2, 5, 10, 11, or 49 lacking the C-terminal 1, 2, 3, 4, 5 or 10 to15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at theN-terminus. A preferred polypeptide will comprise a truncation relativeto SEQ ID NO: 2 or 5 of between 2 and 5 amino acids at the N-terminusand no more than 3 amino acids at the C-terminus. A preferred GDF trapfor use in such a preparation consists of, or consists essentially of,the amino acid sequence of SEQ ID NO: 36.

Optionally, a GDF trap comprising an altered ActRII ligand-bindingdomain has a ratio of K_(d) for activin A binding to K_(d) for GDF11and/or GDF8 binding that is at least 2-, 5-, 10-, 20, 50-, 100-, or even1000-fold greater relative to the ratio for the wild-type ligand-bindingdomain. Optionally, the GDF trap comprising an altered ligand-bindingdomain has a ratio of IC₅₀ for inhibiting activin A to IC₅₀ forinhibiting GDF11 and/or GDF8 that is at least 2-, 5-, 10-, 20-, 25-50-,100-, or even 1000-fold greater relative to the wild-type ActRIIligand-binding domain. Optionally, the GDF trap comprising an alteredligand-binding domain inhibits GDF11 and/or GDF8 with an IC₅₀ at least2, 5, 10, 20, 50, or even 100 times less than the IC₅₀ for inhibitingactivin A. These GDF traps can be fusion proteins that include animmunoglobulin Fc domain (either wild-type or mutant). In certain cases,the subject soluble GDF traps are antagonists (inhibitors) of GDF8and/or GDF11.

In certain aspects, the disclosure provides GDF traps which are solubleActRIIB polypeptides comprising an altered ligand-binding (e.g.,GDF11-binding) domain. GDF traps with altered ligand-binding domains maycomprise, for example, one or more mutations at amino acid residues suchas E37, E39, R40, K55, R56, Y60, A64, K74, W78, L79, D80, F82 and F101of human ActRIIB (numbering is relative to SEQ ID NO: 1). Optionally,the altered ligand-binding domain can have increased selectivity for aligand such as GDF8/GDF11 relative to a wild-type ligand-binding domainof an ActRIIB receptor. To illustrate, these mutations are demonstratedherein to increase the selectivity of the altered ligand-binding domainfor GDF11 (and therefore, presumably, GDF8) over activin: K74Y, K74F,K74I, L79D, L79E, and D801. The following mutations have the reverseeffect, increasing the ratio of activin binding over GDF11: D54A, K55A,L79A and F82A. The overall (GDF11 and activin) binding activity can beincreased by inclusion of the “tail” region or, presumably, anunstructured linker region, and also by use of a K74A mutation. Othermutations that caused an overall decrease in ligand binding affinity,include: R40A, E37A, R56A, W78A, D80K, D80R, D80A, D80G, D80F, D80M andD80N. Mutations may be combined to achieve desired effects. For example,many of the mutations that affect the ratio of GDF11:activin bindinghave an overall negative effect on ligand binding, and therefore, thesemay be combined with mutations that generally increase ligand binding toproduce an improved binding protein with ligand selectivity. In anexemplary embodiment, a GDF trap is an ActRIIB polypeptide comprising anL79D or L79E mutation, optionally in combination with additional aminoacid substitutions, additions, or deletions.

In certain embodiments, ActRII antagonists to be used in accordance withthe methods disclosed herein are ActRIIB polypeptides or ActRIIB-basedGDF trap polypeptides. In general such ActRIIB polypeptides andActRIIB-based GDF trap polypeptides are soluble polypeptides thatcomprise a portion/domain derived from the ActRIIB sequence of SEQ IDNO:1, 4, or 49, particularly an extracellular, ligand-bindingportion/domain derived from the ActRIIB sequence of SEQ ID NO:1, 4, or49. In some embodiments, the portion derived from ActRIIB corresponds toa sequence beginning at any one of amino acids 21-29 (e.g., 21, 22, 23,24, 25, 26, 27, 28, or 29) of SEQ ID NO:1 or 4 [optionally beginning at22-25 (e.g., 22, 23, 24, or 25) of SEQ ID NO:1 or 4] and ending at anyone of amino acids 109-134 (e.g., 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4. In some embodiments,the portion derived from ActRIIB corresponds to a sequence beginning atany one of amino acids 20-29 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28,or 29) of SEQ ID NO: 1 or 4 [optionally beginning at 22-25 (e.g., 22,23, 24, or 25) of SEQ ID NO:1 or 4] and ending at any one of amino acids109-133 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133)of SEQ ID NO: 1 or 4. In some embodiments, the portion derived fromActRIIB corresponds to a sequence beginning at any one of amino acids20-24 (e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 [optionallybeginning at 22-25 (e.g., 22, 23, 24, or 25) of SEQ ID NO:1 or 4] andending at any one of amino acids 109-133 (e.g., 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4. In someembodiments, the portion derived from ActRIIB corresponds to a sequencebeginning at any one of amino acids 21-24 (e.g., 21, 22, 23, or 24) ofSEQ ID NO: 1 or 4 and ending at any of amino acids 109-134 (e.g., 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO:1 or 4. In some embodiments, the portion derived from ActRIIBcorresponds to a sequence beginning at any one of amino acids 20-24(e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any oneof amino acids 118-133 (e.g., 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4 In someembodiments, the portion derived from ActRIIB corresponds to a sequencebeginning at any one of amino acids 21-24 (e.g., 21, 22, 23, or 24) ofSEQ ID NO: 1 or 4 and ending at any one of amino acids 118-134 (e.g.,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, or 134) of SEQ ID NO: 1 or 4. In some embodiments, the portionderived from ActRIIB corresponds to a sequence beginning at any one ofamino acids 20-24 (e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 andending at any one of amino acids 128-133 (e.g., 128, 129, 130, 131, 132,or 133) of SEQ ID NO: 1 or 4. In some embodiments, the portion derivedfrom ActRIIB corresponds to a sequence beginning at any of amino acids20-24 (e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 39 and ending atany of amino acids 128-133 (e.g., 128, 129, 130, 131, 132, or 133) ofSEQ ID NO: 1 or 39. In some embodiments, the portion derived fromActRIIB corresponds to a sequence beginning at any one of amino acids21-29 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4and ending at any one of amino acids 118-134 (e.g., 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) ofSEQ ID NO: 1 or 4. In some embodiments, the portion derived from ActRIIBcorresponds to a sequence beginning at any one of amino acids 20-29(e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4and ending at any one of amino acids 118-133 (e.g., 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ IDNO: 1 or 4. In some embodiments, the portion derived from ActRIIBcorresponds to a sequence beginning at one any of amino acids 21-29(e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4 andending at any one of amino acids 128-134 (e.g., 128, 129, 130, 131, 132,133, or 134) of SEQ ID NO: 1 or 4. In some embodiments, the portionderived from ActRIIB corresponds to a sequence beginning at any one ofamino acids 20-29 (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) ofSEQ ID NO: 1 or 4 and ending at any one of amino acids 128-133 (e.g.,128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4. Surprisingly,ActRIIB and ActRIIB-based GDF trap constructs beginning at 22-25 (e.g.,22, 23, 24, or 25) of SEQ ID NO: 1 or 4 have activity levels greaterthan proteins having the full extracellular domain of human ActRIIB In apreferred embodiment, the ActRIIB polypeptides and ActRIIB-based GDFtrap polypeptides comprise, consist essentially of, or consist of, anamino acid sequence beginning at amino acid position 25 of SEQ ID NO: 1or 4 and ending at amino acid position 131 of SEQ ID NO: 1 or 4. Any ofthe foregoing ActRIIB polypeptides or ActRIIB-based GDF trappolypeptides may be produced as a homodimer. Any of the foregoingActRIIB polypeptides or ActRIIB-based GDF trap polypeptides may furthercomprise a heterologous portion that comprises a constant region from anIgG heavy chain, such as an Fc domain. Any of the above ActRIIB-basedGDF trap polypeptides may comprise an acidic amino acid at the positioncorresponding to position 79 of SEQ ID NO: 1, optionally in combinationwith one or more additional amino acid substitutions, deletions, orinsertions relative to SEQ ID NO: 1. Any of the above ActRIIBpolypeptides or ActRIIB-based GDF trap polypeptides, including homodimerand/or fusion proteins thereof, may bind to and/or inhibit signaling byactivin (e.g., activin A, activin B, activin C, or activin AB) in acell-based assay. Any of the above ActRIIB polypeptides or ActRIIB-basedGDF trap polypeptides, including homodimer and/or fusion proteinsthereof, may bind to and/or inhibit signaling by GDF11 and/or GDF8 in acell based assay. Optionally, any of the above ActRIIB polypeptides orActRIIB-based GDF trap polypeptides, including homodimer and/or fusionproteins thereof, may bind to and/or inhibit signaling of one or more ofactivin B, activin C, activin E, BMP6, BMP7, and Nodal in a cell-basedassay.

Other ActRIIB polypeptides and ActRIIB-based GDF Trap polypeptides arecontemplated, such as the following. An ActRIIB polypeptide or GDF trappolypeptide comprising an amino acid sequence that is at least 80%(e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to thesequence of amino acids 29-109 of SEQ ID NO: 1 or 4, wherein theposition corresponding to 64 of SEQ ID NO: 1 is an R or K, and whereinthe ActRIIB polypeptide or ActRIIB-based GDF trap polypeptide inhibitssignaling by activin, GDF8, and/or GDF11 in a cell-based assay. TheActRIIB polypeptide or ActRIIB-based GDF trap polypeptide as above,wherein at least one alteration with respect to the sequence of SEQ IDNO: 1 or 4 is positioned outside of the ligand-binding pocket. TheActRIIB polypeptide or ActRIIB-based GDF trap polypeptide as above,wherein at least one alteration with respect to the sequence of SEQ IDNO: 1 or 4 is a conservative alteration positioned within theligand-binding pocket. The ActRIIB polypeptide or ActRIIB-based GDF trappolypeptide as above, wherein at least one alteration with respect tothe sequence of SEQ ID NO: 1 or 4 is an alteration at one or morepositions selected from the group consisting of K74, R40, Q53, K55, F82,and L79.

Other ActRIIB polypeptides and ActRIIB-based GDF trap polypeptides arecontemplated, such as the following. An ActRIIB polypeptide orActRIIB-based GDF trap polypeptide comprising an amino acid sequencethat is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 4,and wherein the protein comprises at least one N-X-S/T sequence at aposition other than an endogenous N-X-S/T sequence of ActRIIB, and at aposition outside of the ligand binding pocket. The ActRIIB polypeptideor ActRIIB-based GDF trap polypeptide as above, wherein the ActRIIBpolypeptide or ActRIIB-based GDF trap polypeptide comprises an N at theposition corresponding to position 24 of SEQ ID NO: 1 or 4 and an S or Tat the position corresponding to position 26 of SEQ ID NO: 1 or 4, andwherein the ActRIIB polypeptide or ActRIIB-based GDF trap polypeptideinhibits signaling by activin, GDF8, and/or GDF11 in a cell-based assay.The ActRIIB polypeptide or ActRIIB-based GDF trap polypeptide as above,wherein the ActRIIB polypeptide or ActRIIB-based GDF Trap polypeptidecomprises an R or K at the position corresponding to position 64 of SEQID NO: 1 or 4. The ActRIIB polypeptide or ActRIIB-based GDF trappolypeptide as above, wherein ActRIIB polypeptide or ActRIIB-based GDFtrap polypeptide comprises a D or E at the position corresponding toposition 79 of SEQ ID NO: 1 or 4, and wherein the ActRIIB polypeptide orActRIIB-based GDF trap polypeptide inhibits signaling by activin, GDF8,and/or GDF11 in a cell-based assay. The ActRIIB polypeptide orActRIIB-based GDF trap polypeptide as above, wherein at least onealteration with respect to the sequence of SEQ ID NO: 1 or 4 is aconservative alteration positioned within the ligand-binding pocket. TheActRIIB polypeptide or ActRIIB-based GDF trap polypeptide as above,wherein at least one alteration with respect to the sequence of SEQ IDNO: 1 or 4 is an alteration at one or more positions selected from thegroup consisting of K74, R40, Q53, K55, F82, and L79. The ActRIIBpolypeptide or ActRIIB-based GDF trap polypeptide above, wherein theActRIIB polypeptide or ActRIIB-based GDF trap polypeptide is a fusionprotein further comprising one or more heterologous portion. Any of theabove ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides, orfusion proteins thereof, may be produced as a homodimer. Any of theabove ActRIIB fusion proteins or ActRIIB-based GDF trap fusion proteinsmay have a heterologous portion that comprises a constant region from anIgG heavy chain, such as an Fc domain.

In certain embodiments, a preferred ActRIIB polypeptide, for use inaccordance with the methods disclosed herein, comprises an amino acidsequence that comprises, consists of, or consists essentially of, theamino acid sequence of SEQ ID NOs: 2, 3, 5, 6, 29, 31, or 49, andpolypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to any of the foregoing. An ActRIIB polypeptide may include afunctional fragment of a natural ActRIIB polypeptide, such as onecomprising at least 10, 20 or 30 amino acids of a sequence selected fromSEQ ID NOs: 2, 3, 5, 6, 29, 31, or 49 or a sequence of SEQ ID NO: 2 or5, lacking the C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids andlacking 1, 2, 3, 4 or 5 amino acids at the N-terminus. A preferredpolypeptide will comprise a truncation relative to SEQ ID NO: 2 or 5 ofbetween 2 and 5 amino acids at the N-terminus and no more than 3 aminoacids at the C-terminus. A preferred GDF trap for use in accordance withthe methods described herein consists of, or consists essentially of,the amino acid sequence of SEQ ID NO:29.

A general formula for an active (e.g., ligand binding) ActRIIApolypeptide is one that comprises a polypeptide that starts at aminoacid 30 and ends at amino acid 110 of SEQ ID NO:9. Accordingly, ActRIIApolypeptides and ActRIIA-based GDF traps of the present disclosure maycomprise, consist, or consist essentially of a polypeptide that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aminoacids 30-110 of SEQ ID NO:9. Optionally, ActRIIA polypeptides andActRIIA-based GDF trap polypeptides of the present disclosure comprise,consists, or consist essentially of a polypeptide that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsamino acids 12-82 of SEQ ID NO:9 optionally beginning at a positionranging from 1-5 (e.g., 1, 2, 3, 4, or 5) or 3-5 (e.g., 3, 4, or 5) andending at a position ranging from 110-116 (e.g., 110, 111, 112, 113,114, 115, or 116) or 110-115 (e.g., 110, 111, 112, 113, 114, or 115) orSEQ ID NO:9, respectively, and comprising no more than 1, 2, 5, 10 or 15conservative amino acid changes in the ligand binding pocket, and zero,one or more non-conservative alterations at positions 40, 53, 55, 74, 79and/or 82 in the ligand-binding pocket with respect to SEQ ID NO:9. Anyof the foregoing ActRIIA polypeptides or ActRIIA-based GDF trappolypeptides may be produced as a homodimer. Any of the foregoingActRIIA polypeptides or ActRIIA-based GDF trap polypeptides may furthercomprise a heterologous portion that comprises a constant region from anIgG heavy chain, such as an Fc domain. Any of the above ActRIIApolypeptides or ActRIIA-based GDF trap polypeptides, including homodimerand/or fusion proteins thereof, may bind to and/or inhibit signaling byactivin (e.g., activin A, activin B, activin C, or activin AB) in acell-based assay. Any of the above ActRIIA polypeptides or ActRIIA-basedGDF trap polypeptides, including homodimer and/or fusion proteinsthereof, may bind to and/or inhibit signaling by GDF11 and/or GDF8 in acell-based assay. Optionally, any of the above ActRIIA polypeptides orActRIIB-based GDF trap polypeptides, including homodimer and/or fusionproteins thereof, may bind to and/or inhibit signaling of one or more ofactivin B, activin C, activin E, GDF7, and Nodal in a cell-based assay.

In certain embodiments, preferred ActRIIA polypeptides and ActRIIA-basedGDF-trap polypeptides, for use in accordance with the methods disclosedherein, comprise an amino acid sequence that comprises, consists of, orconsists essentially of, the amino acid sequence of SEQ ID NOs: 9, 10,22, 26, or 28, and polypeptides that are at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to any of the foregoing. An ActRIIApolypeptide or ActRIIA-based GDF-trap polypeptide may include afunctional fragment of a natural ActRIIA polypeptide, such as onecomprising at least 10, 20 or 30 amino acids of a sequence selected fromSEQ ID NOs: 9, 10, 22, 26, or 28 or a sequence of SEQ ID NO:10, lackingthe C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2,3, 4 or 5 amino acids at the N-terminus. A preferred polypeptide willcomprise a truncation relative to SEQ ID NO:10 of between 2 and 5 aminoacids at the N-terminus and no more than 3 amino acids at theC-terminus. A preferred ActRIIA polypeptide for use in the methodsdescribed herein consists of, or consists essentially of, the amino acidsequence of SEQ ID NO: 26 or 28.

An ActRII polypeptide (e.g. an ActRIIA or ActRIIB polypeptide) or GDFtrap polypeptide of the disclosure may include one or more alterations(e.g., amino acid additions, deletions, substitutions, or combinationsthereof) in the amino acid sequence of an ActRII polypeptide (e.g., inthe ligand-binding domain) relative to a naturally occurring ActRIIpolypeptide. The alteration in the amino acid sequence may, for example,alter glycosylation of the polypeptide when produced in a mammalian,insect, or other eukaryotic cell or alter proteolytic cleavage of thepolypeptide relative to the naturally occurring ActRII polypeptide.

Optionally, ActRII polypeptides (e.g. an ActRIIA or ActRIIBpolypeptides) and GDF trap polypeptides of the disclosure comprise oneor more modified amino acid residues selected from: a glycosylated aminoacid, a PEGylated amino acid, a farnesylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, and an amino acid conjugated to an organic derivatizingagent.

In some embodiments, an ActRII polypeptide (e.g. an ActRIIA or ActRIIBpolypeptide) or GDF trap polypeptide of the disclosure may be a fusionprotein that has, as one domain, an ActRII polypeptide or GDF trappolypeptide (e.g., a ligand-binding domain of an ActRII receptor,optionally with one or more sequence variations) and one or moreadditional heterologous domains that provide a desirable property, suchas improved pharmacokinetics, easier purification, targeting toparticular tissues, etc. For example, a domain of a fusion protein mayenhance one or more of in vivo stability, in vivo half-life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, multimerization of the fusion protein, and/orpurification. ActRII polypeptide and GDF trap fusion proteins mayinclude an immunoglobulin Fc domain (wild-type or mutant) or a serumalbumin. In certain embodiments, an ActRII polypeptide and GDF trapfusion protein comprises a relatively unstructured linker positionedbetween the Fc domain and the ActRII or GDF trap domain. Thisunstructured linker may correspond to the roughly 15 amino acidunstructured region at the C-terminal end of the extracellular domain ofActRII or GDF trap (the “tail”), or it may be an artificial sequence ofbetween 3 and 5, 15, 20, 30, 50 or more amino acids that are relativelyfree of secondary structure. A linker may be rich in glycine and prolineresidues and may, for example, contain repeating sequences ofthreonine/serine and glycines [e.g., TG₄ (SEQ ID NO:52), TG₃ (SEQ IDNO:53), or SG₄ (SEQ ID NO:54) singlets or repeats] or a series of threeglycines. A fusion protein may include a purification subsequence, suchas an epitope tag, a FLAG tag, a polyhistidine sequence, and a GSTfusion. In certain embodiments, an ActRII fusion protein or GDF trapfusion comprises a leader sequence. The leader sequence may be a nativeActRII leader sequence (e.g., a native ActRIIA or ActRIIB leadersequence) or a heterologous leader sequence. In certain embodiments, theleader sequence is a tissue plasminogen activator (TPA) leader sequence.In some embodiment, an ActRII fusion protein or GDF trap fusion proteincomprises an amino acid sequence as set forth in the formula A-B-C. TheB portion is an N- and C-terminally truncated ActRII or GDF trappolypeptide as described herein. The A and C portions may beindependently zero, one, or more than one amino acids, and both A and Cportions are heterologous to B. The A and/or C portions may be attachedto the B portion via a linker sequence.

Optionally, ActRII polypeptides (e.g., ActRIIA and ActRIIB polypeptides)or GDF trap polypeptides, including variants and fusion proteinsthereof, to be used in accordance with the methods disclosed herein bindto one or more ActRIIB ligands (e.g., activin A, activin B, activin AB,activin C, activin E, GDF11, GDF8, BMP6, BMP7, and/or Nodal) with a Kdless than 10 micromolar, less than 1 micromolar, less than 100nanomolar, less than 10 nanomolar, or less than 1 nanomolar. Optionally,such ActRII polypeptides or GDF trap polypeptides inhibit ActRIIsignaling, such as ActRIIA and/or ActRIIB intracellular signaltransduction events triggered by an ActRII ligand (e.g., SMAD 2/3 and/orSMAD 1/5/8 signaling).

In certain aspects, the disclosure provides pharmaceutical preparationscomprising an ActRII antagonist of the present disclosure (e.g., anActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap polypeptide) anda pharmaceutically acceptable carrier. A pharmaceutical preparation mayalso include one or more additional compounds such as a compound that isused to treat a disorder or condition described herein (e.g., anadditional compound that increase red blood cell levels in a subject inneed thereof, treat or prevent an anemia in a subject in need thereof(including, e.g., reduction of transfusion burden), treat MDS orsideroblastic anemias in a subject in need thereof, and/or treat orprevent one or more complications of MDS or sideroblastic anemias (e.g.,anemia, blood transfusion requirement, neutropenia, iron overload, acutemyocardial infarction, hepatic failure, hepatomegaly, splenomegaly,progression to acute myeloid lymphoma) and or treat or prevent adisorder associated with SF3B1 mutations in a subject in need thereof).Preferably, a pharmaceutical preparation of the disclosure issubstantially pyrogen-free. In general, it is preferable that an ActRIIApolypeptide, an ActRIIB polypeptide, or a GDF trap polypeptide beexpressed in a mammalian cell line that mediates suitably naturalglycosylation of the polypeptide so as to diminish the likelihood of anunfavorable immune response in a patient. Human and CHO cell lines havebeen used successfully, and it is expected that other common mammalianexpression vectors will be useful. In some embodiments, preferableActRIIA polypeptides, ActRIIB polypeptides, and GDF trap polypeptidesare glycosylated and have a glycosylation pattern that is obtainablefrom a mammalian cell, preferably a CHO cell. In certain embodiments,the disclosure provides packaged pharmaceuticals comprising apharmaceutical preparation described herein and labeled for use in oneor more of increasing red blood cell levels and/or hemoglobin in amammal (preferably a human), treating or preventing anemia in a mammal(preferably a human), treating sideroblastic anemia or MDS in a mamamal(preferably a human), and/or treating or preventing one or morecomplications of sideroblastic anemia or MDS (e.g., anemia,vaso-occlusive crisis, etc.) in a mammal (preferably a human) or treator prevent a disorder associated with SF3B1 mutations in a mammal(preferably a human).

In certain aspects, the disclosure provides nucleic acids encoding anActRII polypeptide (e.g., an ActRIIA or ActRIIB polypeptide) or GDF trappolypeptide. An isolated polynucleotide may comprise a coding sequencefor a soluble ActRII polypeptide or GDF trap polypeptide, such asdescribed herein. For example, an isolated nucleic acid may include asequence coding for an ActRII polypeptide or GDF trap comprising anextracellular domain (e.g., ligand-binding domain) of an ActRIIpolypeptide having one or more sequence variations and a sequence thatwould code for part or all of the transmembrane domain and/or thecytoplasmic domain of an ActRII polypeptide, but for a stop codonpositioned within the transmembrane domain or the cytoplasmic domain, orpositioned between the extracellular domain and the transmembrane domainor cytoplasmic domain. For example, an isolated polynucleotide codingfor a GDF trap may comprise a full-length ActRII polynucleotide sequencesuch as SEQ ID NO: 1, 4, or 9 or having one or more variations, or apartially truncated version, said isolated polynucleotide furthercomprising a transcription termination codon at least six hundrednucleotides before the 3′-terminus or otherwise positioned such thattranslation of the polynucleotide gives rise to an extracellular domainoptionally fused to a truncated portion of a full-length ActRII. Nucleicacids disclosed herein may be operably linked to a promoter forexpression, and the disclosure provides cells transformed with suchrecombinant polynucleotides. Preferably the cell is a mammalian cell,such as a CHO cell.

In certain aspects, the disclosure provides methods for making an ActRIIpolypeptide or a GDF trap. Such a method may include expressing any ofthe nucleic acids disclosed herein (e.g., SEQ ID NO: 8, 13, 27, 32, 39,42, 46, or 48) in a suitable cell, such as a Chinese hamster ovary (CHO)cell. Such a method may comprise: a) culturing a cell under conditionssuitable for expression of the GDF trap polypeptide, wherein said cellis transformed with a GDF trap expression construct; and b) recoveringthe GDF trap polypeptide so expressed. GDF trap polypeptides may berecovered as crude, partially purified or highly purified fractionsusing any of the well-known techniques for obtaining protein from cellcultures.

In certain aspects, the present disclosure relates to an antibody, orcombination of antibodies, that antagonize ActRII activity (e.g.,inhibition of ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 and/or SMAD 1/5/8 signaling). In particular, the disclosureprovides methods of using an antibody ActRII antagonist, or combinationof antibody ActRII antagonists, to, e.g., increase red blood cell levelsin a subject in need thereof, treat or prevent an anemia in a subject inneed thereof (including, e.g., reduction of transfusion burden), treatMDS or sideroblastic anemias in a subject in need thereof, and/or treator prevent one or more complications of MDS or sideroblastic anemias(e.g., anemia, blood transfusion requirement, neutropenia, ironoverload, acute myocardial infarction, hepatic failure, hepatomegaly,splenomegaly, progression to acute myeloid lymphoma) and or treat orprevent a disorder associated with SF3B1, DNMT3A, and/or TET2 mutationsin a subject in need thereof.

In certain embodiments, a preferred antibody ActRII antagonist of thedisclosure is an antibody, or combination of antibodies, that binds toand/or inhibits activity of at least GDF11 (e.g., GDF11-mediatedactivation of ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 signaling). Optionally, the antibody, or combination ofantibodies, further binds to and/or inhibits activity of GDF8 (e.g.,GDF8-mediated activation of ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling), particularly in the case of amultispecific antibody that has binding affinity for both GDF11 and GDF8or in the context of a combination of one or more anti-GDF11 antibodyand one or more anti-GDF8 antibody. Optionally, an antibody, orcombination of antibodies, of the disclosure does not substantially bindto and/or inhibit activity of activin A (e.g., activin A-mediatedactivation of ActRIIA or ActRIIB signaling transduction, such as SMAD2/3 signaling). In some embodiments, an antibody, or combination ofantibodies, of the disclosure that binds to and/or inhibits the activityof GDF11 and/or GDF8 further binds to and/or inhibits activity of one ofmore of activin A, activin B, activin AB, activin C, activin E, BMP6,BMP7, and Nodal (e.g., activation of ActRIIA or ActRIIB signalingtransduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling),particularly in the case of a multispecific antibody that has bindingaffinity for multiple ActRII ligands or in the context of a combinationmultiple antibodies—each having binding affinity for a different ActRIIligand.

In part, the disclosure demonstrates that ActRII antagonists may be usedin combination (e.g., administered at the same time or different times,but generally in such a manner as to achieve overlapping pharmacologicaleffects) with EPO receptor activators to increase red blood cell levels(erythropoiesis) or, as examples, treat or prevent an anemia in asubject in need thereof (including, e.g., reduction of transfusionburden), treat MDS or sideroblastic anemias in a subject in needthereof, and/or treat or prevent one or more complications of MDS orsideroblastic anemias (e.g., anemia, blood transfusion requirement,neutropenia, iron overload, acute myocardial infarction, hepaticfailure, hepatomegaly, splenomegaly, progression to acute myeloidlymphoma) and or treat or prevent a disorder associated with SF3B1,DNMT3A, and/or TET2 mutations in a subject in need thereof. In part, thedisclosure demonstrates that a GDF trap can be administered incombination with an EPO receptor activator to synergistically increaseformation of red blood cells in a patient, particularly in sideroblasticanemia or MDS patients. Thus, the effect of this combined treatment canbe significantly greater than the sum of the effects of the ActRIIantagonists and the EPO receptor activator when administered separatelyat their respective doses. In certain embodiments, this synergism may beadvantageous since it enables target levels of red blood cells to beattained with lower doses of an EPO receptor activator, thereby avoidingpotential adverse effects or other problems associated with higherlevels of EPO receptor activation. Accordingly, in certain embodiments,the methods of the present disclosure (e.g., methods of increase redblood cell levels in a subject in need thereof, treat or prevent ananemia in a subject in need thereof (including, e.g., reduction oftransfusion burden), treat MDS or sideroblastic anemias in a subject inneed thereof, and/or treat or prevent one or more complications of MDSor sideroblastic anemias (e.g., anemia, blood transfusion requirement,neutropenia, iron overload, acute myocardial infarction, hepaticfailure, hepatomegaly, splenomegaly, progression to acute myeloidlymphoma) and or treat or prevent a disorder associated with SF3B1,DNMT3A, and/or TET2 mutations in a subject in need thereof) compriseadministering a patient in need thereof one or more ActRII antagonists(e.g., ActRIIA polypeptides, ActRIIB polypeptides, and/or GDF trappolypeptides) in combination with one or more EPO receptor activators.

An EPO receptor activator may stimulate erythropoiesis by directlycontacting and activating EPO receptor. In certain embodiments, the EPOreceptor activator is one of a class of compounds based on the 165amino-acid sequence of native EPO and generally known aserythropoiesis-stimulating agents (ESAs), examples of which are epoetinalfa, epoetin beta, epoetin delta, and epoetin omega. In otherembodiments, ESAs include synthetic EPO proteins (SEPs) and EPOderivatives with nonpeptidic modifications conferring desirablepharmacokinetic properties (lengthened circulating half-life), examplesof which are darbepoetin alfa and methoxy-polyethylene-glycol epoetinbeta. In certain embodiments, an EPO receptor activator may be an EPOreceptor agonist that does not incorporate the EPO polypeptide backboneor is not generally classified as an ESA. Such EPO receptor agonists mayinclude, but are not limited to, peptidic and nonpeptidic mimetics ofEPO, agonistic antibodies targeting EPO receptor, fusion proteinscomprising an EPO mimetic domain, and erythropoietin receptorextended-duration limited agonists (EREDLA).

In certain embodiments, an EPO receptor activator may stimulateerythropoiesis indirectly, without contacting EPO receptor itself, byenhancing production of endogenous EPO. For example, hypoxia-inducibletranscription factors (HIFs) are endogenous stimulators of EPO geneexpression that are suppressed (destabilized) under normoxic conditionsby cellular regulatory mechanisms. In part, the disclosure providesincreased erythropoiesis in a patient by combined treatment with a GDFtrap and an indirect EPO receptor activator with HIF stabilizingproperties, such as a prolyl hydroxylase inhibitor.

In certain instances, when administering a GDF trap polypeptide forthese other therapeutic indications, it may be desirable to monitor theeffects on red blood cells during administration of the ActRIIantagonist, or to determine or adjust the dosing of the ActRIIantagonist, in order to reduce undesired effects on red blood cells. Forexample, increases in red blood cell levels, hemoglobin levels, orhematocrit levels may cause increases in blood pressure.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or patent application filed contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1 shows an alignment of extracellular domains of human ActRIIA (SEQID NO: 56) and human ActRIIB (SEQ ID NO: 2) with the residues that arededuced herein, based on composite analysis of multiple ActRIIB andActRIIA crystal structures, to directly contact ligand indicated withboxes.

FIG. 2 shows a multiple sequence alignment of various vertebrate ActRIIBproteins and human ActRIIA (SEQ ID NOs: 57-64).

FIG. 3 shows the purification of ActRIIA-hFc expressed in CHO cells. Theprotein purifies as a single, well-defined peak as visualized by sizingcolumn (top panel) and Coomassie stained SDS-PAGE (bottom panel) (leftlane: molecular weight standards; right lane: ActRIIA-hFc).

FIG. 4 shows the binding of ActRIIA-hFc to activin and GDF-11, asmeasured by Biacore™ assay.

FIGS. 5A and 5B shows the effects of ActRIIA-hFc on red blood cellcounts in female non-human primates (NHPs). Female cynomolgus monkeys(four groups of five monkeys each) were treated with placebo or 1 mg/kg,10 mg/kg or 30 mg/kg of ActRIIA-hFc on day 0, day 7, day 14, and day 21.FIG. 5A shows red blood cell (RBC) counts. FIG. 5B shows hemoglobinlevels. Statistical significance is relative to baseline for eachtreatment group. At day 57, two monkeys remained in each group.

FIGS. 6A and 6B shows the effects of ActRIIA-hFc on red blood cellcounts in male non-human primates. Male cynomolgus monkeys (four groupsof five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg, or30 mg/kg of ActRIIA-hFc on day 0, day 7, day 14, and day 21. FIG. 6Ashows red blood cell (RBC) counts. FIG. 6B shows hemoglobin levels.Statistical significance is relative to baseline for each treatmentgroup. At day 57, two monkeys remained in each group.

FIGS. 7A and 7B shows the effects of ActRIIA-hFc on reticulocyte countsin female non-human primates. Cynomolgus monkeys (four groups of fivemonkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg, or 30mg/kg of ActRIIA-hFc on day 0, day 7, day 14, and day 21. FIG. 7A showsabsolute reticulocyte counts. FIG. 7B shows the percentage ofreticulocytes relative to RBCs. Statistical significance is relative tobaseline for each group. At day 57, two monkeys remained in each group.

FIGS. 8A and 8B shows the effects of ActRIIA-hFc on reticulocyte countsin male non-human primates. Cynomolgus monkeys (four groups of fivemonkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg, or 30mg/kg of ActRIIA-hFc on day 0, day 7, day 14, and day 21. FIG. 8A showsabsolute reticulocyte counts. FIG. 8B shows the percentage ofreticulocytes relative to RBCs. Statistical significance is relative tobaseline for each group. At day 57, two monkeys remained in each group.

FIG. 9 shows results from the human clinical trial described in Example5, where the area-under-curve (AUC) and administered dose of ActRIIA-hFchave a linear correlation, regardless of whether ActRIIA-hFc wasadministered intravenously (IV) or subcutaneously (SC).

FIG. 10 shows a comparison of serum levels of ActRIIA-hFc in patientsadministered IV or SC.

FIG. 11 shows bone alkaline phosphatase (BAP) levels in response todifferent dose levels of ActRIIA-hFc. BAP is a marker for anabolic bonegrowth.

FIG. 12 depicts the median change from baseline of hematocrit levelsfrom the human clinical trial described in Example 5. ActRIIA-hFc wasadministered intravenously (IV) at the indicated dosage.

FIG. 13 depicts the median change from baseline of hemoglobin levelsfrom the human clinical trial described in Example 5. ActRIIA-hFc wasadministered intravenously (IV) at the indicated dosage.

FIG. 14 depicts the median change from baseline of RBC (red blood cell)count from the human clinical trial described in Example 5. ActRIIA-hFcwas administered intravenously (IV) at the indicated dosage.

FIG. 15 depicts the median change from baseline of reticulocyte countfrom the human clinical trial described in Example 5. ActRIIA-hFc wasadministered intravenously (IV) at the indicated dosage.

FIG. 16 shows the full amino acid sequence for the GDF trap ActRIIB(L79D20-134)-hFc (SEQ ID NO:38), including the TPA leader sequence (doubleunderlined), ActRIIB extracellular domain (residues 20-134 in SEQ ID NO:1; underlined), and hFc domain. The aspartate substituted at position 79in the native sequence is double underlined and highlighted, as is theglycine revealed by sequencing to be the N-terminal residue in themature fusion protein.

FIGS. 17A and 17B show a nucleotide sequence encoding ActRIIB(L79D20-134)-hFc. SEQ ID NO:39 corresponds to the sense strand, and SEQ IDNO:40 corresponds to the antisense strand. The TPA leader (nucleotides1-66) is double underlined, and the ActRIIB extracellular domain(nucleotides 76-420) is underlined.

FIG. 18 shows the full amino acid sequence for the truncated GDF trapActRIIB(L79D 25-131)-hFc (SEQ ID NO:41), including the TPA leader(double underlined), truncated ActRIIB extracellular domain (residues25-131 in SEQ ID NO:1; underlined), and hFc domain. The aspartatesubstituted at position 79 in the native sequence is double underlinedand highlighted, as is the glutamate revealed by sequencing to be theN-terminal residue in the mature fusion protein.

FIGS. 19A and 19B shows a nucleotide sequence encoding ActRIIB(L79D25-131)-hFc. SEQ ID NO:42 corresponds to the sense strand, and SEQ IDNO:43 corresponds to the antisense strand. The TPA leader (nucleotides1-66) is double underlined, and the truncated ActRIIB extracellulardomain (nucleotides 76-396) is underlined. The amino acid sequence forthe ActRIIB extracellular domain (residues 25-131 in SEQ ID NO: 1) isalso shown.

FIG. 20 shows the amino acid sequence for the truncated GDF trapActRIIB(L79D 25-131)-hFc without a leader (SEQ ID NO:44). The truncatedActRIIB extracellular domain (residues 25-131 in SEQ ID NO:1) isunderlined. The aspartate substituted at position 79 in the nativesequence is double underlined and highlighted, as is the glutamaterevealed by sequencing to be the N-terminal residue in the mature fusionprotein.

FIG. 21 shows the amino acid sequence for the truncated GDF trapActRIIB(L79D 25-131) without the leader, hFc domain, and linker (SEQ IDNO:45). The aspartate substituted at position 79 in the native sequenceis underlined and highlighted, as is the glutamate revealed bysequencing to be the N-terminal residue in the mature fusion protein.

FIGS. 22A and 22B shows an alternative nucleotide sequence encodingActRIIB(L79D 25-131)-hFc. SEQ ID NO:46 corresponds to the sense strand,and SEQ ID NO:47 corresponds to the antisense strand. The TPA leader(nucleotides 1-66) is double underlined, the truncated ActRIIBextracellular domain (nucleotides 76-396) is underlined, andsubstitutions in the wild-type nucleotide sequence of the extracellulardomain are double underlined and highlighted (compare with SEQ ID NO:42,FIGS. 19A and 19B). The amino acid sequence for the ActRIIBextracellular domain (residues 25-131 in SEQ ID NO:1) is also shown.

FIG. 23 shows nucleotides 76-396 (SEQ ID NO:48) of the alternativenucleotide sequence shown in FIGS. 22A and 22B (SEQ ID NO:46). The samenucleotide substitutions indicated in FIGS. 22A and 22B are alsounderlined and highlighted here. SEQ ID NO:48 encodes only the truncatedActRIIB extracellular domain (corresponding to residues 25-131 in SEQ IDNO:1) with a L79D substitution, e.g., ActRIIB(L79D 25-131).

FIG. 24 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change in redblood cell concentration from baseline in cynomolgus monkey.VEH=vehicle. Data are means±SEM. n=4-8 per group.

FIG. 25 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change inhematocrit from baseline in cynomolgus monkey. VEH=vehicle. Data aremeans±SEM. n=4-8 per group.

FIG. 26 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change inhemoglobin concentration from baseline in cynomolgus monkey.VEH=vehicle. Data are means±SEM. n=4-8 per group.

FIG. 27 shows the effect of treatment with ActRIIB(L79D 20-134)-hFc(gray) or ActRIIB(L79D 25-131)-hFc (black) on the absolute change incirculating reticulocyte concentration from baseline in cynomolgusmonkey. VEH=vehicle. Data are means±SEM. n=4-8 per group.

FIG. 28 shows the effect of combined treatment with erythropoietin (EPO)and ActRIIB(L79D 25-131)-hFc for 72 hours on hematocrit in mice. Dataare means±SEM (n=4 per group), and means that are significantlydifferent from each other (p<0.05, unpaired t-test) are designated bydifferent letters. Combined treatment increased hematocrit by 23%compared to vehicle, a synergistic increase greater than the sum of theseparate effects of EPO and ActRIIB(L79D 25-131)-hFc.

FIG. 29 shows the effect of combined treatment with EPO and ActRIIB(L79D25-131)-hFc for 72 hours on hemoglobin concentrations in mice. Data aremeans±SEM (n=4 per group), and means that are significantly differentfrom each other (p<0.05) are designated by different letters. Combinedtreatment increased hemoglobin concentrations by 23% compared tovehicle, which was also a synergistic effect.

FIG. 30 shows the effect of combined treatment with EPO and ActRIIB(L79D25-131)-hFc for 72 hours on red blood cell concentrations in mice. Dataare means±SEM (n=4 per group), and means that are significantlydifferent from each other (p<0.05) are designated by different letters.Combined treatment increased red blood cell concentrations by 20%compared to vehicle, which was also a synergistic effect.

FIG. 31 shows the effect of combined treatment with EPO and ActRIIB(L79D25-131)-hFc for 72 hours on numbers of erythropoietic precursor cells inmouse spleen. Data are means±SEM (n=4 per group), and means that aresignificantly different from each other (p<0.01) are designated bydifferent letters. Whereas EPO alone increased the number of basophilicerythroblasts (BasoE) dramatically at the expense of late-stageprecursor maturation, combined treatment increased BasoE numbers to alesser but still significant extent while supporting undiminishedmaturation of late-stage precursors.

FIG. 32 shows that a GDF trap can mitigate ineffective erythropoiesisand ameliorate anemia at multiple stages of disease severity in a mousemodel of MDS. (A) RBC numbers and hemoglobin concentrations (top) andmorphological enumeration of hematopoietic precursors in bone marrow(bottom) in wild-type (Wt) mice treated with vehicle (Tris-bufferedsaline, TBS, n=5), MDS mice treated with TBS (n=5), and MDS mice treatedwith ActRIIB(L79D 25-131)-mFc (RAP-536, 10 mg/kg, n=6) twice weekly for8 weeks ending at approximately 6 months of age (early stage). *P<0.05,**P<0.01, vs. TBS-treated MDS mice; ^(###)P<0.001 vs. wild-type mice.(B) Same endpoints as in panel A in MDS mice treated with RAP-536 (10mg/kg, twice weekly, n=5) or TBS (n=4) for 7 weeks ending atapproximately 12 months of age (late stage). *P<0.05 vs. TBS-treated MDSmice. Data are means±SEM.

DETAIL DESCRIPTION OF THE INVENTION 1. Overview

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. By manipulating theactivity of a member of the TGF-beta family, it is often possible tocause significant physiological changes in an organism. For example, thePiedmontese and Belgian Blue cattle breeds carry a loss-of-functionmutation in the GDF8 (also called myostatin) gene that causes a markedincrease in muscle mass [see, e.g., Grobet et al. (1997) Nat Genet.17(1):71-4]. Furthermore, in humans, inactive alleles of GDF8 areassociated with increased muscle mass and, reportedly, exceptionalstrength [see, e.g., Schuelke et al. (2004) N Engl J Med, 350:2682-8].

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream SMAD proteins (e.g., SMAD proteins 1, 2, 3, 5, and 8) uponligand stimulation [see, e.g., Massagué (2000) Nat. Rev. Mol. Cell Biol.1:169-178]. These type I and type II receptors are transmembraneproteins, composed of a ligand-binding extracellular domain withcysteine-rich region, a transmembrane domain, and a cytoplasmic domainwith predicted serine/threonine specificity. Type I receptors areessential for signaling. Type II receptors are required for bindingligands and for activation of type I receptors. Type I and II activinreceptors form a stable complex after ligand binding, resulting inphosphorylation of type I receptors by type II receptors.

Two related type II receptors (ActRII), ActRIIA and ActRIIB, have beenidentified as the type II receptors for activins [see, e.g., Mathews andVale (1991) Cell 65:973-982; and Attisano et al. (1992) Cell 68:97-108]. Besides activins, ActRIIA and ActRIIB can biochemicallyinteract with several other TGF-β family proteins including, forexample, BMP6, BMP7, Nodal, GDF8, and GDF11 [see, e.g., Yamashita et al.(1995) J. Cell Biol. 130:217-226; Lee and McPherron (2001) Proc. Natl.Acad. Sci. USA 98:9306-9311; Yeo and Whitman (2001) Mol. Cell 7:949-957; and Oh et al. (2002) Genes Dev. 16:2749-54]. ALK4 is theprimary type I receptor for activins, particularly for activin A, andALK-7 may serve as a receptor for other activins as well, particularlyfor activin B. In certain embodiments, the present disclosure relates toantagonizing a ligand of an ActRII receptor (also referred to as anActRII ligand) with one or more inhibitor agents disclosed herein,particularly inhibitor agents that can antagonize one or more of activinA, activin B, activin C, activin E, GDF11 and/or GDF8.

Activins are dimeric polypeptide growth factors that belong to theTGF-beta superfamily. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B), respectively). The human genomealso encodes an activin C and an activin E, which are primarilyexpressed in the liver, and heterodimeric forms containing β_(C) orβ_(E) are also known.

In the TGF-beta superfamily, activins are unique and multifunctionalfactors that can stimulate hormone production in ovarian and placentalcells, support neuronal cell survival, influence cell-cycle progresspositively or negatively depending on cell type, and induce mesodermaldifferentiation at least in amphibian embryos [DePaolo et al. (1991)Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997) Curr Biol.7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963]. Moreover,erythroid differentiation factor (EDF) isolated from the stimulatedhuman monocytic leukemic cells was found to be identical to activin A[Murata et al. (1988) PNAS, 85:2434]. It has been suggested that activinA promotes erythropoiesis in the bone marrow. In several tissues,activin signaling is antagonized by its related heterodimer, inhibin.For example, during the release of follicle-stimulating hormone (FSH)from the pituitary, activin promotes FSH secretion and synthesis, whileinhibin prevents FSH secretion and synthesis. Other proteins that mayregulate activin bioactivity and/or bind to activin include follistatin(FS), follistatin-related protein (FSRP, also known as FLRG or FSTL3),and α₂-macroglobulin.

As described herein, agents that bind to “activin A” are agents thatspecifically bind to the β_(A) subunit, whether in the context of anisolated β_(A) subunit or as a dimeric complex (e.g., a PAPA homodimeror a β_(A)β_(B) heterodimer). In the case of a heterodimer complex(e.g., a β_(A)β_(B) heterodimer), agents that bind to “activin A” arespecific for epitopes present within the PA subunit, but do not bind toepitopes present within the non-β_(A) subunit of the complex (e.g., theβ_(B) subunit of the complex). Similarly, agents disclosed herein thatantagonize (inhibit) “activin A” are agents that inhibit one or moreactivities as mediated by a PA subunit, whether in the context of anisolated PA subunit or as a dimeric complex (e.g., a PAPA homodimer or aβ_(A)β_(B) heterodimer). In the case of β_(A)β_(B) heterodimers, agentsthat inhibit “activin A” are agents that specifically inhibit one ormore activities of the PA subunit, but do not inhibit the activity ofthe non-β_(A) subunit of the complex (e.g., the β_(B) subunit of thecomplex). This principle applies also to agents that bind to and/orinhibit “activin B”, “activin C”, and “activin E”. Agents disclosedherein that antagonize “activin AB” are agents that inhibit one or moreactivities as mediated by the β_(A) subunit and one or more activitiesas mediated by the β_(B) subunit.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as SMAD proteins. Studiessupport the idea that ActRIIA and ActRIIB serve as type II receptors forNodal [see, e.g., Sakuma et al. (2002) Genes Cells. 2002, 7:401-12]. Itis suggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate SMAD2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway SMADs, SMAD2 and SMAD3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and differentiation factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle [McPherron et al., Nature (1997)387:83-90]. Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle [see, e.g., Ashmore etal. (1974) Growth, 38:501-507; Swatland and Kieffer (1994) J. Anim. Sci.38:752-757; McPherron and Lee (1997) Proc. Natl. Acad. Sci. USA94:12457-12461; and Kambadur et al. (1997) Genome Res. 7:910-915] and,strikingly, in humans [see, e.g., Schuelke et al. (2004) N Engl J Med350:2682-8]. Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression [see, e.g., Gonzalez-Cadavid et al. (1998) PNAS 95:14938-43].In addition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation [see,e.g. international patent application publication no. WO 00/43781]. TheGDF8 propeptide can noncovalently bind to the mature GDF8 domain dimer,inactivating its biological activity [see, e.g., Miyazono et al. (1988)J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol. Chem.,263: 7646-7654; and Brown et al. (1990) Growth Factors, 3: 35-43]. Otherproteins which bind to GDF8 or structurally related proteins and inhibittheir biological activity include follistatin, and potentially,follistatin-related proteins [see, e.g., Gamer et al. (1999) Dev. Biol.,208: 222-232].

Growth and differentiation factor-11 (GDF11), also known as BMP11, is asecreted protein [McPherron et al. (1999) Nat. Genet. 22: 260-264].GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development [see, e.g.,Nakashima et al. (1999) Mech. Dev. 80: 185-189]. GDF11 plays a uniquerole in patterning both mesodermal and neural tissues [see, e.g., Gameret al. (1999) Dev Biol., 208:222-32]. GDF11 was shown to be a negativeregulator of chondrogenesis and myogenesis in developing chick limb[see, e.g., Gamer et al. (2001) Dev Biol. 229:407-20]. The expression ofGDF11 in muscle also suggests its role in regulating muscle growth in asimilar way to GDF8. In addition, the expression of GDF11 in brainsuggests that GDF11 may also possess activities that relate to thefunction of the nervous system. Interestingly, GDF11 was found toinhibit neurogenesis in the olfactory epithelium [see, e.g., Wu et al.(2003) Neuron. 37:197-207].

Bone morphogenetic protein (BMP7), also called osteogenic protein-1(OP-1), is well known to induce cartilage and bone formation. Inaddition, BMP7 regulates a wide array of physiological processes. Forexample, BMP7 may be the osteoinductive factor responsible for thephenomenon of epithelial osteogenesis. It is also found that BMP7 playsa role in calcium regulation and bone homeostasis. Like activin, BMP7binds to type II receptors, ActRIIA and ActRIIB However, BMP7 andactivin recruit distinct type I receptors into heteromeric receptorcomplexes. The major BMP7 type I receptor observed was ALK2, whileactivin bound exclusively to ALK4 (ActRIIB) BMP7 and activin eliciteddistinct biological responses and activated different SMAD pathways[see, e.g., Macias-Silva et al. (1998) J Biol Chem. 273:25628-36].

As demonstrated herein, ActRII polypeptides (e.g., ActRIIA and ActRIIBpolypeptides) can be used to increase red blood cell levels in vivo. Incertain examples, it is shown that a GDF trap polypeptide (specificallya variant ActRIIB polypeptide) is characterized by unique biologicalproperties in comparison to a corresponding sample of a wild-type(unmodified) ActRII polypeptide. This GDF trap is characterized, inpart, by substantial loss of binding affinity for activin A, andtherefore significantly diminished capacity to antagonize activin Aactivity, but retains near wild-type levels of binding and inhibition ofGDF11. In vivo, the GDF trap is more effective at increasing red bloodcell levels as compared to the wild-type ActRIIB polypeptide and hasbeneficial effects in a variety of models for anemia. It should be notedthat hematopoiesis is a complex process, regulated by a variety offactors, including erythropoietin, G-CSF, and iron homeostasis. Theterms “increase red blood cell levels” and “promote red blood cellformation” refer to clinically observable metrics, such as hematocrit,red blood cell counts, and hemoglobin measurements, and are intended tobe neutral as to the mechanism by which such changes occur.

The data of the present disclosure therefore indicate that the observedbiological activity of an ActRII polypeptide, with respect to red bloodcell levels, is not dependent on activin A inhibition. However, it is tobe noted that the unmodified ActRIIB polypeptide, which retains activinA binding, still demonstrates the capacity to increase red blood cellsin vivo. Furthermore, an ActRIIB or ActRIIA polypeptide that retainsactivin A inhibition may be more desirable in some applications, incomparison to a GDF trap having diminished binding affinity for activinA, where more modest gains in red blood cell levels are desirable and/orwhere some level of off-target activity is acceptable (or evendesirable).

Accordingly, the methods of the present disclosure, in general, aredirected to the use of one or more ActRII antagonist agents describedherein, optionally in combination with one or more supportive therapies,to increase red blood cell formation in a subject in need thereof, treator prevent an anemia in a subject in need thereof, to treatmyelodysplastic syndrome, to treat sideroblastic anemia in a subject inneed thereof, and to treat or prevent one or more complications ofsideroblastic anemia or myelodysplastic syndrome (e.g., anemia, bloodtransfusion requirement, iron overload, neutropenia, splenomegaly,progression to acute myeloid leukemia), and, optionally, in a subgroupof patients with ring sideroblasts and/or one or more mutations in theSF3B1, DNMT3A, and/or TET2 gene in bone marrow cells. Another subgroupof patients that are identified as being particularly likely to respondto an ActRII antagonist is patients that have failed prior treatmentwith EPO therapy or other EPO receptor activator therapy.

As evidenced herein, the ActRII antagonist agents described may be usedin combination with an EPO receptor activator or in patients that havefailed treatment with EPO receptor activators. EPO is a glycoproteinhormone involved in the growth and maturation of erythroid progenitorcells into erythrocytes. EPO is produced by the liver during fetal lifeand by the kidney in adults. Decreased production of EPO, which commonlyoccurs in adults as a consequence of renal failure, leads to anemia. EPOhas been produced by genetic engineering techniques based on expressionand secretion of the protein from a host cell transfected with the EPOgene. Administration of such recombinant EPO has been effective in thetreatment of anemia. For example, Eschbach et al. (1987, N Engl J Med316:73) describe the use of EPO to correct anemia caused by chronicrenal failure.

Effects of EPO are mediated through its binding to, and activation of, acell surface receptor belonging to the cytokine receptor superfamily anddesignated the EPO receptor. The human and murine EPO receptors havebeen cloned and expressed [see, e.g., D'Andrea et al. (1989) Cell57:277; Jones et al. (1990) Blood 76:31; Winkelman et al. (1990) Blood76:24; and U.S. Pat. No. 5,278,065]. The human EPO receptor gene encodesa 483-amino-acid transmembrane protein comprising an extracellulardomain of approximately 224 amino acids and exhibits approximately 82%amino acid sequence identity with the murine EPO receptor (see, e.g.,U.S. Pat. No. 6,319,499). The cloned, full-length EPO receptor expressedin mammalian cells (66-72 kDa) binds EPO with an affinity (K_(D)=100-300nM) similar to that of the native receptor on erythroid progenitorcells. Thus, this form is thought to contain the main EPO bindingdeterminant and is referred to as the EPO receptor. By analogy withother closely related cytokine receptors, the EPO receptor is thought todimerize upon agonist binding. Nevertheless, the detailed structure ofthe EPO receptor, which may be a multimeric complex, and its specificmechanism of activation are not completely understood (see, e.g., U.S.Pat. No. 6,319,499).

Activation of the EPO receptor results in several biological effects.These include increased proliferation of immature erythroblasts,increased differentiation of immature erythroblasts, and decreasedapoptosis in erythroid progenitor cells [see, e.g., Liboi et al. (1993)Proc Natl Acad Sci USA 90:11351-11355; Koury et al. (1990) Science248:378-381]. The EPO receptor signal transduction pathways mediatingproliferation and differentiation appear to be distinct [see, e.g.,Noguchi et al. (1988) Mol Cell Biol 8:2604; Patel et al. (1992) J BiolChem, 267:21300; and Liboi et al. (1993) Proc Natl Acad Sci USA90:11351-11355]. Some results suggest that an accessory protein may berequired for mediation of the differentiation signal [see, e.g., Chibaet al. (1993) Nature 362:646; and Chiba et al. (1993) Proc Natl Acad SciUSA 90:11593]. However, there is controversy regarding the role ofaccessory proteins in differentiation since a constitutively activatedform of the receptor can stimulate both proliferation anddifferentiation [see, e.g., Pharr et al. (1993) Proc Natl Acad Sci USA90:938].

EPO receptor activators include small moleculeerythropoiesis-stimulating agents (ESAs) as well as EPO-based compounds.An example of the former is a dimeric peptide-based agonist covalentlylinked to polyethylene glycol (proprietary names Hematide™ andOmontys®), which has shown erythropoiesis-stimulating properties inhealthy volunteers and in patients with both chronic kidney disease andendogenous anti-EPO antibodies [see, e.g., Stead et al. (2006) Blood108:1830-1834; and Macdougall et al. (2009) N Engl J Med 361:1848-1855].Other examples include nonpeptide-based ESAs [see, e.g., Qureshi et al.(1999) Proc Natl Acad Sci USA 96:12156-12161].

EPO receptor activators also include compounds that stimulateerythropoiesis indirectly, without contacting EPO receptor itself, byenhancing production of endogenous EPO. For example, hypoxia-inducibletranscription factors (HIFs) are endogenous stimulators of EPO geneexpression that are suppressed (destabilized) under normoxic conditionsby cellular regulatory mechanisms. Therefore, inhibitors of HIF prolylhydroxylase enzymes are being investigated for EPO-inducing activity invivo. Other indirect activators of EPO receptor include inhibitors ofGATA-2 transcription factor [see, e.g., Nakano et al. (2004) Blood104:4300-4307], which tonically inhibits EPO gene expression, andinhibitors of hemopoietic cell phosphatase (HCP or SHP-1), whichfunctions as a negative regulator of EPO receptor signal transduction[see, e.g., Klingmuller et al. (1995) Cell 80:729-738].

As described herein, patients that exhibit ring sideroblasts may beparticularly suited to treatment with ActRII antagonists. Sideroblasticanemias can be classified broadly into congenital (inherited) andacquired forms, which can be further subdivided as shown in Table 1.

TABLE 1 Classification of Sideroblastic Anemias* Class Gene AnemiaSeverity Iron Homeostasis Congenital Nonsyndromic X-linked ALAS2 Mild tosevere Iron overload SLC25A38 deficiency SLC25A38 Severe Iron overloadGlutaredoxin 5 deficiency GLRX5 Mild to severe Iron overloadErythropoietic protoporphyria FECH Mild — Syndromic X-linked with ataxiaABCB7 Mild to moderate — SIFD Unknown Severe Iron overload Pearsonmarrow- pancreas mtDNA Severe — Syndrome Myopathy, lactic acidosis, andPUS1/YARS2 Mild to severe — sideroblastic anemia (MLASA)Thiamine-responsive SLC19A2 Severe — megaloblastic anemia (TRMA)Syndromic/nonsyndromic of Unknown Variable — unknown cause AcquiredClonal/Neoplastic MDS** Variable Mild to severe Iron overload MetabolicAlcoholism — Variable — Drug-induced — Variable — Copper deficiency(zinc toxicity) — Variable — Hypothermia — Variable — *See Bottomley etal., 2014, Hematol Oncol Clin N Am 28: 653-670. **See table below forMDS subclassifications according to the World Health Organization.

MDS represent the most common class of acquired bone-marrow-failuresyndromes in adults. Although MDS are increasingly well understood froma biological standpoint, improved pathologic insight has not yettranslated into highly effective or curative therapies for most patientssuffering from these disorders. Increasing failure of cellulardifferentiation in MDS is associated with evolution to secondary acutemyeloid leukemia (AML), which is currently defined by the WHO as havingat least 20% myeloblasts in the blood or marrow, or the presence of oneof several AML-defining karyotypic abnormalities regardless of blastproportion [see, e.g., Vardiman et al. (2009) Blood 114:937-951]. AML isultimately diagnosed in as many as 30% of MDS patients. Since thebiological heterogeneity that underlies MDS translates into widevariations in clinical outcomes, prognostic classification schemes havebeen developed to predict the natural course of MDS and to counselpatients [Zeidan et al (2013) Curr Hematol Malig Rep 8:351-360]. TheInternational Prognostic Scoring System (IPSS) is an example of one suchclassification that categorizes patients according to risk profile,ranging from Low (median survival of 5.7 years) to Intermediate 1(median survival of 3.5 years) to Intermediate 2 (median survival of 1.2years) to High (median survival of 0.4 years). An alternate systemreferred to as IPSS-R may also be used for patient stratification. Froma patient's perspective, the prognosis helps define the severity ofdisease and sets expectations as to how it is likely to impact them.From a physician's standpoint, the prognosis provides a means of stagingthe disease in a manner that can be used to help direct therapy.Notably, such schemes do not typically take patient comorbidities intoaccount and are not intended to predict clinical benefit in relation toa specific therapy.

Additional MDS classification systems have been proposed to facilitateappropriate treatment and management of patients with MDS.Classification has evolved over several decades to incorporate progressin understanding these complex syndromes. The French-American-British(FAB) classification scheme for MDS was proposed in 1982 [Bennett et al.(1982) Br J Haematol 51:189-199] and served as the basis for a modifiedclassification system established by the WHO in 2001. As summarized inTable 2 below, the current version of the WHO classification (revised in2008) is based on (1) the percentage of myeloblasts in the bone marrowand peripheral blood, (2) the type and degree of dysplasia, (3) thepresence of ring sideroblasts, and (4) the presence of cytogeneticabnormalities. Thus, ring sideroblasts are characteristic of RARS butcan also be present in other subtypes of MDS [see, e.g., Juneja et al.(1983) J Clin Pathol 36:566-569; Malcovati et al. (2013) Best Pract ResClin Haematol 26:377-385]. Depending on the MDS subtype, anemia canoccur, for example, in the presence or absence of ring sideroblasts,alone or in combination with abnormally low numbers of neutrophils(neutropenia), low numbers of platelets (thrombocytopenia), or elevatedlevels of platelets (thrombocytosis). Refractory anemia with ringsideroblasts associated with marked thrombocytosis (RARS-T) is currentlyincluded as a provisional entry in the WHO classification (Table 2)within the group of MDS neoplasms unclassifiable (MDS-U). RARS-T isdefined as anemia with dysplastic ineffective erythropoiesis and ringsideroblasts≥15% of erythroid precursors, no blasts in peripheral bloodand <5% in the bone marrow, and thrombocytosis with a plateletcount≥450×10⁹/L [Malcovati et al. (2013) Best Pract Res Clin Haematol26:377-385]. Due to a compromised immune system, patients withneutropenia may be at serious risk of infection and even sepsis, and itis therefore important to treat this condition. Patients withthrombocytopenia are at increased risk of internal hemorrhage, anddepending on severity it may also be beneficial to treat this condition.

TABLE 2 2008 WHO Classification System for MDS* MDS Subtype BloodFindings Bone Marrow Findings Refractory cytopenia with Predominantlyunicytopenia Unilineage dysplasia: ≥10% of cells in one myeloidunilineage dysplasia (RCUD): lineage (a) refractory anemia, (b) <5%blasts refractory neutropenia, or (c) <15% of erythroid precursors arering sideroblasts refractory thrombocytopenia Refractory anemia withring Anemia ≥15% of erythroid precursors are ring sideroblastssideroblasts (RARS) No blasts Erythroid dysplasia only <5% blastsRefractory cytopenia with Cytopenia(s) Dysplasia in ≥10% of cells in ≥2myeloid lineages multilineage dysplasia No or rare blasts (<1%)(neutrophil and/or erythroid precursors and/or (RCMD) No Auer rodsmegakaryocytes) <1 × 10⁹ per L monocytes <5% blasts No Auer rods ±15%ring sideroblasts Refractory anemia with excess Cytopenia(s) Unilineageor multilineage dysplasia blasts-1 (RAEB-1) <5% blasts 5%-9% blasts NoAuer rods No Auer rods <1 × 10⁹ per L monocytes Refractory anemia withexcess Cytopenia(s) Unilineage or multilineage dysplasia blasts-1(RAEB-2) 5%-19% blasts 10%-19% blasts Auer rods± Auer rods± <1 × 10⁹ perL monocytes Myelodysplastic syndrome- Cytopenias Unequivocal dysplasiain <10% of cells in one or more unclassified (MDS-U) ** <1% blastsmyeloid lineages when accompanied by a cytogenetic abnormalityconsidered as presumptive evidence for a diagnosis of MDS <5% blasts MDSassociated with isolated Anemia Normal-to-increased megakaryocytes withhypolobated del(5q) Usually normal or increased nuclei platelet count<5% blasts No or rare blasts (<1%) Isolated del(5q) cytogeneticabnormality No Auer rods *From Vardiman et al (2009) Blood 114: 937-951** Includes refractory anemia with ring sideroblasts associated withmarked thrombocytosis (RARS-T)

In one embodiment of the disclosure, ActRII antagonists are useful fortreating anemia in patients, including MDS patients or patients withsideroblastic anemia, in whom more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% oferythroid precursors are ring sideroblasts, e.g., in refractory anemiawith ring sideroblasts (RARS), RARS associated with markedthrombocytosis (RARS-T), or refractory cytopenia with multilineagedysplasia (RCMD, also known as RCMD-RS in patients where ringsideroblasts are prominent).

Anemia occurs frequently in MDS. Approximately 80% of MDS patientspresent with anemia, and a substantial percentage of them becomedependent on blood transfusions during the course of their disease[Steensma et al. (2006) Mayo Clin Proc 81:104-130]. Some MDS subtypesare characterized by “ineffective erythropoiesis”, in which there isimpaired differentiation (maturation) of late-stage erythroid precursorcells despite EPO-stimulated hyperproliferation of early-stage erythroidprogenitor cells in response to tissue hypoxia. Thus, a key sign ofineffective erythropoiesis is persistent anemia in spite of elevatedlevels of endogenous EPO. Ineffective erythropoiesis occurs frequentlyin the RARS subtype of MDS but not the RAEB subtype, which ischaracterized by relative hypoproliferation of the erythroid marrow[Cazzola et al. (1982) Br J Haematol 50:55-62]. Circulating levels ofhepcidin, a critical regulator of iron homeostasis, are about an orderof magnitude lower in RARS than RAEB [Santini et al. (2011) PLoS One6:e23109]. Since low levels of hepcidin promote iron absorption, theinappropriately low levels of hepcidin measured in disorders such asRARS and thalassemia are thought to account for iron overload observedin these disorders even in the absence of blood transfusions.

Chronic red blood cell transfusions alleviate anemia but expose patientsto multiple risks, including infectious disease, allergic or hemolyticreactions, and exacerbation of iron overload [Rawn (2008) Curr OpinAnaesthesiol 21:664-668; Ozcan et al. (2013) Expert Rev Hematol6:165-189]. As systemic iron levels increase, the body increasesferritin production for iron storage and reduces transferrin receptorproduction to reduce iron entry into cells. When the iron-bindingcapacity of circulating transferrin is exceeded, iron is found in theplasma as non-transferrin bound iron (NTBI). In MDS, levels ofnon-transferrin bound iron are higher in low-risk than high-risksubtypes and highest in RARS [Santini et al. (2011) PLoS One 6:e23109].Since iron cannot be actively secreted from the body, it initiallyaccumulates in the reticuloendothelial macrophages and is laterdeposited primarily in parenchymal cells of the heart, liver, andendocrine glands [Siah et al. (2006) Clin Biochem Rev 27:5-16]. Underconditions of iron overload, non-transferrin bound iron changes to itsredox-active form known as labile plasma iron, which is transported intocells where it promotes formation of reactive oxygen species. Thesehighly toxic molecules adversely impact hematopoiesis and particularlydamage cardiac, hepatic, and endocrine tissues.

The MDS patient population consists mainly of elderly with comorbidconditions—including a propensity for cardiac failure, infection,hemorrhage, and hepatic cirrhosis—and iron overload may rapidlyexacerbate such pre-existing conditions. Compared to MDS patients athigh risk of developing AML, patients at low or intermediate-1 risk maybe more prone to iron overload due to their longer life expectancy. Forthese reasons, iron chelation therapy to reduce iron burden isconsidered advisable in patients with low- or intermediate-1 risk MDSsubtypes who have a long life expectancy and are anticipated to receivemore than 20 RBC transfusions [Temraz et al. (2014) Crit Rev OncolHematol 91:64-73].

Novel sequencing techniques have led in the past few years toidentification of dozens of genes that are recurrently mutated in MDS. A2013 list of such genes classified by type is shown in Table 3. One ormore such mutations can be found in almost all patients with MDS, andknowing the nature of the genes involved has improved understanding ofhow MDS develops and evolves, although it has not yet had an impact ontreatment. Whole-genome sequencing applied to MDS patient samples hasidentified an entirely novel class of cancer-associated genes encodingmRNA splicing (spliceosome) factors. The first such gene identified inMDS was SF3B1, which is mutated particularly frequently in patients withRARS [Papaemmanuil et al. (2011) N Engl J Med 365:1384-1395]. Othermajor categories of mutated genes are epigenetic (DNA methylation)regulators, transcription factors, and signaling molecules [Cazzola etal. (2013) Blood 122:4021-4034; Bejar et al. (2014) Blood124:2793-2803]. The extent to which these mutations co-occur in MDSpatients seems to vary with gene type. For example, approximately 50% ofMDS patients possess one of ten genes identified to date encoding mutantsplicing factors, but these mutant genes rarely co-occur in the samepatient [Bejar et al. (2014) Blood 124:2793-2803]. Thus, these mutantgenes are seldom redundant markers for the same individuals. Genesencoding mutant epigenetic regulators co-occur more frequently with eachother and with mutant splicing factor genes in the same patient. Asdisclosed herein, the differential occurrence of mutant genes such asthose listed in Table 3 provides a genetic signature that can assist inpredicting which patients with MDS or sideroblastic anemia are likely tobe either responsive or nonresponsive therapeutically to an ActRIIantagonist.

TABLE 3 MDS-Associated Somatic Mutations* Frequency in MDS Gene (%cases) RNA Splicing SF3B1 14-28 SRSF2 15 U2AF1  8 ZRSR2  6 PRPF40B  1SF3A1  1 SF1  1 U2AF65 <1 LUC7L2 Rare PRPF8 Rare Epigenetic RegulatorsTET2 19-26 ASXL1 10-20 DNMT3A 10 IDH1/IDH2  4-12 EZH2  6 UTX  1 ATRX <1Transcription Factors RUNX1 10-20 TP53  4-14 ETV6 1-3 PHF6 Rare WT1 RareSignaling NRAS 10 CBL  3 JAK2  3 FLT3 2-3 KRAS 1-2 c-KIT  1 BRAF <1CDKN2A <1 GNAS <1 PTEN <1 PTPN11 <1 CBLB Rare MPL, CSF1R Rare OthersNPM1 2-3 *From Tothova et al. (2013) Clin Cancer Res 19: 1637-1643.

Among the genes listed in Table 3, the gene encoding splicing factor 3B1(SF3B1) has been implicated recently as critical in MDS, particularly inthe RARS, RARS-T, and RCMD-RS subtypes [Malcovati et al. (2011) Blood118:6239-6246; Dolatshad et al. (2014) Leukemia doi:10.1038/leu.2014.331 epub ahead of print]. Somatic mutations in SF3B1also occur in hematologic cancers including chronic lymphocytic leukemia(CLL), and acute myeloid leukemia (AML) as well as in breast cancer,pancreatic cancer, gastric cancer, prostate cancer, and uveal melanoma[Malcovati et al. (2011) Blood 118:6239-6246; Wang et al. (2011) N EnglJ Med 365:2497-2506; The Cancer Genome Atlas Network (2012) Nature490:61-70; Biankin et al. (2012) Nature 491:399-405; Chesnais et al.(2012) Oncotarget 3:1284-1293; Furney et al. (2013) Cancer Discov3:1122-1129; Je et al. (2013) Int J Cancer 133:260-266]. A spectrum ofSF3B1 mutations, many clustered at a few locations in the protein, havebeen identified in clinical samples or in cell lines exposed to highconcentrations of pladienolide [Webb et al. (2013) Drug Discov Today18:43-49]. SF3B1 mutations identified in MDS include, for example,K182E, E491G, R590K, E592K, R625C, R625G, N626D, N626S, H662Y, T663A,K666M, K666Q, K666R, Q670E, G676D, V701I, I704N, I704V, G740R, A744P,D781G, and A1188V. SF3B1 mutations identified in cancer include, forexample, N619K, N626H, N626Y, R630S, I704T, G740E, K741N, G742D, D894G,Q903R, R1041H, and I1241T. Finally, SF3B1 mutations found in both MDSand cancer include, for example, G347V, E622D, Y623C, R625H, R625L,H662D, H662Q, T663I, K666E, K666N, K666T, K700E, and V701F.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of thedisclosure and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which they areused.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalto the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid (nucleic acid) sequenceidentity values are generated using the sequence comparison computerprogram ALIGN-2. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc., and the source code has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

“Agonize”, in all its grammatical forms, refers to the process ofactivating a protein and/or gene (e.g., by activating or amplifying thatprotein's gene expression or by inducing an inactive protein to enter anactive state) or increasing a protein's and/or gene's activity.

“Antagonize”, in all its grammatical forms, refers to the process ofinhibiting a protein and/or gene (e.g., by inhibiting or decreasing thatprotein's gene expression or by inducing an active protein to enter aninactive state) or decreasing a protein's and/or gene's activity.

As used herein, unless otherwise stated, “does not substantially bind toX” is intended to mean that an agent has a K_(D) that is greater thanabout 10⁻⁷, 10 ⁻⁶, 10⁻⁵, 10⁻⁴, or greater (e.g., no detectable bindingby the assay used to determine the K_(D)) for “X” or has relativelymodest binding for “X”, e.g., about 1×10⁻⁸ M or about 1×10⁻⁹ M.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. In general, such interval of accuracy is ±10%. Alternatively, andparticularly in biological systems, the terms “about” and“approximately” may mean values that are within an order of magnitude,preferably ≤5-fold and more preferably ≤2-fold of a given value.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

2. ActRII Antagonists

The data presented herein demonstrates that antagonists (inhibitors) ofActRII (e.g., antagonist of ActRIIA and/or ActRIIB SMAD 2/3 and/or SMAD1/5/8 signaling) can be used to increase red blood cell levels in vivoand provide other benefits to patients. In particular, such ActRIIantagonists are shown herein to be effective in treating various anemiasas well as various complications (e.g., disorders/conditions) of MDS andsideroblastic anemias. Accordingly, the present disclosure provides, inpart, various ActRII antagonist agents that can be used, alone or incombination with one or more erythropoiesis stimulating agents (e.g.,EPO) or other supportive therapies [e.g., hematopoietic growth factors(e.g., G-CSF or GM-CSF), transfusion of red blood cells or whole blood,iron chelation therapy], increase red blood cell levels in a subject inneed thereof, treat or prevent an anemia in a subject in need thereof(including, e.g., reduction of transfusion burden), treat MDS orsideroblastic anemias in a subject in need thereof, and/or treat orprevent one or more complications of MDS or sideroblastic anemias (e.g.,anemia, blood transfusion requirement, neutropenia, iron overload, acutemyocardial infarction, hepatic failure, hepatomegaly, splenomegaly,progression to acute myeloid lymphoma) and or treat or prevent adisorder associated with SF3B1, DNMT3A, and/or TET2 mutations in asubject in need thereof.

In certain embodiments, preferred ActRII antagonists to be used inaccordance with the methods disclosed herein are GDF-ActRII antagonists(e.g., antagonists of GDF-mediated ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling), particularly GDF11- and/orGDF8-mediated ActRII signaling. In some embodiments, preferred ActRIIantagonists of the present disclosure are soluble ActRII polypeptides(e.g., soluble ActRIIA and ActRIIB polypeptides) and GDF trappolypeptides, such as ActRIIA-Fc fusion proteins, ActRIIB-Fc fusionproteins, and GDF trap-Fc fusion proteins.

Although soluble ActRII polypeptides and GDF trap polypeptides of thedisclosure may affect red blood cell levels and/or various complicationsof MDS and sideroblastic anemia through a mechanism other than GDF (e.g.GDF11 and/or GDF8) antagonism [e.g., GDF11 and/or GDF8 inhibition may bean indicator of the tendency of an agent to inhibit the activities of aspectrum of additional agents, including, perhaps, other members of theTGF-beta superfamily (e.g., activin B, activin C, activin E, BMP6, BMP7,and/or Nodal) and such collective inhibition may lead to the desiredeffect on, e.g., hematopoiesis], other types of GDF-ActRII antagonistare expected to be useful including, for example, anti-GDF11 antibodies;anti-GDF8 antibodies; anti-activin A, B, C and/or E antibodies,anti-ActRIIA antibodies; anti-ActRIIB antibodies; anti-ActRIIA/IMantibodies, antisense, RNAi, or ribozyme nucleic acids that inhibit theproduction of one or more of GDF11, GDF8, ActRIIA, and/or ActRIIB; andother inhibitors (e.g., small-molecule inhibitors) of one or more ofGDF11, GDF8, ActRIIA, and/or ActRIIB, particularly agents that disruptGDF11- and/or GDF8-ActRIIA binding and/or GDF11- and/or GDF8-ActRIIBbinding as well as agents that inhibit expression of one or more ofGDF11, GDF8, ActRIIA, and/or ActRIIB Optionally, GDF-ActRII antagonistsof the present disclosure may bind to and/or inhibit the activity (orexpression) of other ActRII ligands including, for example, activin A,activin AB, activin B, activin C, activin E, BMP6, BMP7, and/or Nodal.Optionally, a GDF-ActRII antagonist of the present disclosure may beused in combination with at least one additional ActRII antagonist agentthat binds to and/or inhibits the activity (or expression) of one ormore additional ActRII ligands including, for example, activin A,activin AB, activin B, activin C, activin E, BMP6, BMP7, and/or Nodal.In some embodiments, ActRII antagonists to be used in accordance withthe methods disclosed herein do not substantially bind to and/or inhibitactivin A (e.g., activin A-mediated activation of ActRIIA and/or ActRIIBsignaling transduction, such as SMAD 2/3 signaling).

A. ActRII Polypeptides and GDF Traps

In certain aspects, the present disclosure relates to ActRIIpolypeptides. In particular, the disclosure provides methods of usingActRII polypeptides, alone or in combination with one or moreerythropoiesis stimulating agents (e.g., EPO) or other supportivetherapies [e.g., hematopoietic growth factors (e.g., G-CSF or GM-CSF),transfusion of red blood cells or whole blood, iron chelation therapy],to, e.g., increase red blood cell levels in a subject in need thereof,treat or prevent an anemia in a subject in need thereof (including,e.g., reduction of transfusion burden), treat MDS or sideroblasticanemias in a subject in need thereof, and/or treat or prevent one ormore complications of MDS or sideroblastic anemias (e.g., anemia, bloodtransfusion requirement, neutropenia, iron overload, acute myocardialinfarction, hepatic failure, hepatomegaly, splenomegaly, progression toacute myeloid lymphoma) and or treat or prevent a disorder associatedwith SF3B1, DNMT3A, and/or TET2 mutations in a subject in need thereof.As used herein the term “ActRII” refers to the family of type II activinreceptors. This family includes both the activin receptor type IIA andthe activin receptor type IIB.

As used herein, the term “ActRIIB” refers to a family of activinreceptor type IIB (ActRIIB) proteins from any species and variantsderived from such ActRIIB proteins by mutagenesis or other modification.Reference to ActRIIB herein is understood to be a reference to any oneof the currently identified forms. Members of the ActRIIB family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular domain comprising a cysteine-rich region, a transmembranedomain, and a cytoplasmic domain with predicted serine/threonine kinaseactivity.

The term “ActRIIB polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIB family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRIIA polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNo. WO 2006/012627, which is incorporated herein by reference in itsentirety. Optionally, ActRIIB polypeptides of the present disclosure canbe used to increase red blood cell levels in a subject. Numbering ofamino acids for all ActRIIB-related polypeptides described herein isbased on the numbering of the human ActRIIB precursor protein sequenceprovided below (SEQ ID NO:1), unless specifically designated otherwise.

The human ActRIIB precursor protein sequence is as follows:

(SEQ ID NO: 1) 1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER T NQSGLERCE 51 GEQDKRLHCY ASWR N SSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY 101FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS 151LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR 201FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA 251EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY 301LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK 351PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC 401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL 451AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV 501 TNVDLPPKES SI

The signal peptide is indicated with single underline; the extracellulardomain is indicated in bold font; and the potential, endogenous N-linkedglycosylation sites are indicated with double underline.

The processed soluble (extracellular) human ActRIIB polypeptide sequenceis as follows:

(SEQ ID NO: 2) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT.

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by single underline. The sequence with the “tail”deleted (a 415 sequence) is as follows:

(SEQ ID NO: 3) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHL PEA.

A form of ActRIIB with an alanine at position 64 of SEQ ID NO:1 (A64) isalso reported in the literature [see, e.g., Hilden et al. (1994) Blood,83(8): 2163-2170]. Applicants have ascertained that an ActRIIB-Fc fusionprotein comprising an extracellular domain of ActRIIB with the A64substitution has a relatively low affinity for activin and GDF11. Bycontrast, the same ActRIIB-Fc fusion protein with an arginine atposition 64 (R64) has an affinity for activin and GDF11 in the lownanomolar to high picomolar range. Therefore, sequences with an R64 areused as the “wild-type” reference sequence for human ActRIIB in thisdisclosure.

The form of ActRIIB with an alanine at position 64 is as follows:

(SEQ ID NO: 4) 1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER TNQSGLERCE51 GEQDKRLHCY ASWANSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY 101FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS 151LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR 201FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA 251EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY 301LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK 351PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC 401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL 451AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV 501TNVDLPPKES SI.

The signal peptide is indicated by single underline and theextracellular domain is indicated by bold font.

The processed soluble (extracellular) ActRIIB polypeptide sequence ofthe alternative A64 form is as follows:

(SEQ ID NO: 5) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT.

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by single underline. The sequence with the “tail”deleted (a 415 sequence) is as follows:

(SEQ ID NO: 6) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHL PEA.

The nucleic acid sequence encoding human ActRIIB precursor protein isshown below (SEQ ID NO: 7), consisting of nucleotides 25-1560 of GenbankReference Sequence NM_001106.3, which encode amino acids 1-513 of theActRIIB precursor. The sequence as shown provides an arginine atposition 64 and may be modified to provide an alanine instead. Thesignal sequence is underlined.

(SEQ ID NO: 7) 1 ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC51 CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG 101CCAACTGGGA GCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA 151GGCGAGCAGG ACAAGCGGCT GCACTGCTAC GCCTCCTGGC GCAACAGCTC 201TGGCACCATC GAGCTCGTGA AGAAGGGCTG CTGGCTAGAT GACTTCAACT 251GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCC CCAGGTGTAC 301TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC 351AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA 401CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC 451CTCATCGTCC TGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA 501CGGTCATGTG GACATCCATG AGGACCCTGG GCCTCCACCA CCATCCCCTC 551TGGTGGGCCT GAAGCCACTG CAGCTGCTGG AGATCAAGGC TCGGGGGCGC 601TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTG TAGCTGTCAA 651GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT 701TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC 751GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT 801CCATGACAAG GGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT 851GGAACGAACT GTGTCATGTA GCAGAGACGA TGTCACGAGG CCTCTCATAC 901CTGCATGAGG ATGTGCCCTG GTGCCGTGGC GAGGGCCACA AGCCGTCTAT 951TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAG AGCGACCTCA 1001CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA 1051CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC 1101TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA 1151TTGACATGTA TGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC 1201AAGGCTGCAG ACGGACCCGT GGATGAGTAC ATGCTGCCCT TTGAGGAAGA 1251GATTGGCCAG CACCCTTCGT TGGAGGAGCT GCAGGAGGTG GTGGTGCACA 1301AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACA CCCGGGCCTG 1351GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC 1401TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT 1451CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC 1501ACCAATGTGG ACCTGCCCCC TAAAGAGTCA AGCATC.

A nucleic acid sequence encoding processed soluble (extracellular) humanActRIIB polypeptide is as follows (SEQ ID NO: 8). The sequence as shownprovides an arginine at position 64, and may be modified to provide analanine instead.

(SEQ ID NO: 8) 1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51 GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC 101AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC 151ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA 201TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT 251GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTT GCCAGAGGCT 301GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC.

In certain embodiments, the present disclosure relates to ActRIIApolypeptides. As used herein, the term “ActRIIA” refers to a family ofactivin receptor type IIA (ActRIIA) proteins from any species andvariants derived from such ActRIIA proteins by mutagenesis or othermodification. Reference to ActRIIA herein is understood to be areference to any one of the currently identified forms. Members of theActRIIA family are generally transmembrane proteins, composed of aligand-binding extracellular domain comprising a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ActRIIA polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIA family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRIIA polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNo. WO 2006/012627, which is incorporated herein by reference in itsentirety. Optionally, ActRIIA polypeptides of the present disclosure canbe used to increase red blood cell levels in a subject. Numbering ofamino acids for all ActRIIA-related polypeptides described herein isbased on the numbering of the human ActRIIA precursor protein sequenceprovided below (SEQ ID NO:9), unless specifically designated otherwise.

The human ActRIIA precursor protein sequence is as follows:

(SEQ ID NO: 9) 1 MGAAAKLAFA VFLISCSSGA ILGRSETQEC LFFNANWEKD RT NQTGVEPC 51 YGDKDKRRHC FATWK N ISGS IEIVKQGCWL DDINCYDRTD CVEKKDSPEV 101YFCCCEGNMC NEKFSYFPEM EVTQPTSNPV TPKPPYYNIL LYSLVPLMLI 151AGIVICAFWV YRHHKMAYPP VLVPTQDPGP PPPSPLLGLK PLQLLEVKAR 201GRFGCVWKAQ LLNEYVAVKI FPIQDKQSWQ NEYEVYSLPG MKHENILQFI 251GAEKRGTSVD VDLWLITAFH EKGSLSDFLK ANVVSWNELC HIAETMARGL 301AYLHEDIPGL KDGHKPAISH RDIKSKNVLL KNNLTACIAD FGLALKFEAG 351KSAGDTHGQV GTRRYMAPEV LEGAINFQRD AFLRIDMYAM GLVLWELASR 401CTAADGPVDE YMLPFEEEIG QHPSLEDMQE VVVHKKKRPV LRDYWQKHAG 451MAMLCETIEE CWDHDAEARL SAGCVGERIT QMQRLTNIIT TEDIVTVVTM 501VTNVDFPPKE SSL

The signal peptide is indicated by single underline; the extracellulardomain is indicated in bold font; and the potential, endogenous N-linkedglycosylation sites are indicated by double underline.

The processed soluble (extracellular) human ActRIIA polypeptide sequenceis as follows:

(SEQ ID NO: 10) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP

The C-terminal “tail” of the extracellular domain is indicated by singleunderline. The sequence with the “tail” deleted (a Δ15 sequence) is asfollows:

(SEQ ID NO: 11) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM

The nucleic acid sequence encoding human ActRIIA precursor protein isshown below (SEQ ID NO: 12), as follows nucleotides 159-1700 of GenbankReference Sequence NM_001616.4. The signal sequence is underlined.

(SEQ ID NO: 12) 1 atgggagctg ctgcaaagtt ggcgtttgcc gtctttctta tctcctgttc51 ttcaggtgct atacttggta gatcagaaac tcaggagtgt cttttcttta 101 atgctaattgggaaaaagac agaaccaatc aaactggtgt tgaaccgtgt 151 tatggtgaca aagataaacggcggcattgt tttgctacct ggaagaatat 201 ttctggttcc attgaaatag tgaaacaaggttgttggctg gatgatatca 251 actgctatga caggactgat tgtgtagaaa aaaaagacagccctgaagta 301 tatttttgtt gctgtgaggg caatatgtgt aatgaaaagt tttcttattt351 tccggagatg gaagtcacac agcccacttc aaatccagtt acacctaagc 401caccctatta caacatcctg ctctattcct tggtgccact tatgttaatt 451 gcggggattgtcatttgtgc attttgggtg tacaggcatc acaagatggc 501 ctaccctcct gtacttgttccaactcaaga cccaggacca cccccacctt 551 ctccattact aggtttgaaa ccactgcagttattagaagt gaaagcaagg 601 ggaagatttg gttgtgtctg gaaagcccag ttgcttaacgaatatgtggc 651 tgtcaaaata tttccaatac aggacaaaca gtcatggcaa aatgaatacg701 aagtctacag tttgcctgga atgaagcatg agaacatatt acagttcatt 751ggtgcagaaa aacgaggcac cagtgttgat gtggatcttt ggctgatcac 801 agcatttcatgaaaagggtt cactatcaga ctttcttaag gctaatgtgg 851 tctcttggaa tgaactgtgtcatattgcag aaaccatggc tagaggattg 901 gcatatttac atgaggatat acctggcctaaaagatggcc acaaacctgc 951 catatctcac agggacatca aaagtaaaaa tgtgctgttgaaaaacaacc 1001 tgacagcttg cattgctgac tttgggttgg ccttaaaatt tgaggctggc1051 aagtctgcag gcgataccca tggacaggtt ggtacccgga ggtacatggc 1101tccagaggta ttagagggtg ctataaactt ccaaagggat gcatttttga 1151 ggatagatatgtatgccatg ggattagtcc tatgggaact ggcttctcgc 1201 tgtactgctg cagatggacctgtagatgaa tacatgttgc catttgagga 1251 ggaaattggc cagcatccat ctcttgaagacatgcaggaa gttgttgtgc 1301 ataaaaaaaa gaggcctgtt ttaagagatt attggcagaaacatgctgga 1351 atggcaatgc tctgtgaaac cattgaagaa tgttgggatc acgacgcaga1401 agccaggtta tcagctggat gtgtaggtga aagaattacc cagatgcaga 1451gactaacaaa tattattacc acagaggaca ttgtaacagt ggtcacaatg 1501 gtgacaaatgttgactttcc tcccaaagaa tctagtcta

The nucleic acid sequence encoding processed soluble (extracellular)human ActRIIA polypeptide is as follows:

(SEQ ID NO: 13) 1 atacttggta gatcagaaac tcaggagtgt cttttcttta atgctaattg51 ggaaaaagac agaaccaatc aaactggtgt tgaaccgtgt tatggtgaca 101 aagataaacggcggcattgt tttgctacct ggaagaatat ttctggttcc 151 attgaaatag tgaaacaaggttgttggctg gatgatatca actgctatga 201 caggactgat tgtgtagaaa aaaaagacagccctgaagta tatttttgtt 251 gctgtgaggg caatatgtgt aatgaaaagt tttcttattttccggagatg 301 gaagtcacac agcccacttc aaatccagtt acacctaagc caccc.

An alignment of the amino acid sequences of human ActRIIB solubleextracellular domain and human ActRIIA soluble extracellular domain areillustrated in FIG. 1. This alignment indicates amino acid residueswithin both receptors that are believed to directly contact ActRIIligands. FIG. 2 depicts a multiple-sequence alignment of variousvertebrate ActRIIB proteins and human ActRIIA. From these alignments itis possible to predict key amino acid positions within theligand-binding domain that are important for normal ActRII-ligandbinding activities as well as to predict amino acid positions that arelikely to be tolerant to substitution without significantly alteringnormal ActRII-ligand binding activities.

In other aspects, the present disclosure relates to GDF trappolypeptides (also referred to as “GDF traps”) which may be used, forexample, alone or in combination with one or more erythropoiesisstimulating agents (e.g., EPO) or other supportive therapies [e.g.,hematopoietic growth factors (e.g., G-CSF or GM-CSF), transfusion of redblood cells or whole blood, iron chelation therapy], to, e.g., increasered blood cell levels in a subject in need thereof, treat or prevent ananemia in a subject in need thereof (including, e.g., reduction oftransfusion burden), treat MDS or sideroblastic anemias in a subject inneed thereof, and/or treat or prevent one or more complications of MDSor sideroblastic anemias (e.g., anemia, blood transfusion requirement,neutropenia, iron overload, acute myocardial infarction, hepaticfailure, hepatomegaly, splenomegaly, progression to acute myeloidlymphoma) in a subject in need thereof.

In some embodiments, GDF traps of the present disclosure are soluble,variant ActRII polypeptides (e.g., ActRIIA and ActRIIB polypeptides)that comprise one or more mutations (e.g., amino acid additions,deletions, substitutions, and combinations thereof) in the extracellulardomain (also referred to as the ligand-binding domain) of an ActRIIpolypeptide (e.g., a “wild-type” ActRII polypeptide) such that thevariant ActRII polypeptide has one or more altered ligand-bindingactivities than the corresponding wild-type ActRII polypeptide. Inpreferred embodiments, GDF trap polypeptides of the present disclosureretain at least one similar activity as a corresponding wild-type ActRIIpolypeptide (e.g., an ActRIIA or ActRIIB polypeptide). For example, aGDF trap may bind to and/or inhibit (e.g. antagonize) the function ofone or more ActRII ligands (e.g., inhibit ActRII ligand-mediatedactivation of the ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 and/or SMAD 1/5/8 signaling pathway). In some embodiments, GDFtraps of the present disclosure bind to and/or inhibit one or more ofactivin A, activin B, activin AB, activin C, activin E, Nodal, GDF8,GDF11, BMP6 and/or BMP7).

In certain embodiments, GDF trap polypeptides of the disclosure haveelevated binding affinity for one or more specific ActRII ligands (e.g.,GDF8, GDF11, BMP6, Nodal, and/or BMP7). In other embodiments, GDF trappolypeptides of the disclosure have decreased binding affinity for oneor more specific ActRII ligands (e.g., activin A, activin B, activin AB,activin C, and/or activin E). In still other embodiments, GDF trappolypeptides of the disclosure have elevated binding affinity for one ormore specific ActRII ligands and decreased binding affinity for one ormore different/other ActRII ligands. Accordingly, the present disclosureprovides GDF trap polypeptides that have an altered binding specificityfor one or more ActRII ligands.

In certain preferred embodiments, GDF traps of the present disclosureare designed to preferentially bind to and antagonize GDF11 and/or GDF8(also known as myostatin), e.g., in comparision to a wild-type ActRIIpolypeptide. Optionally, such GDF11 and/or GDF8-binding traps mayfurther bind to and/or antagonize one or more of Nodal, GDF8, GDF11,BMP6 and/or BMP7. Optionally, such GDF11 and/or GDF8-binding traps mayfurther bind to and/or antagonize one or more of activin B, activin C,activin E, Nodal, GDF8, GDF11, BMP6 and/or BMP7. Optionally, such GDF11and/or GDF8-binding traps may further bind to and/or antagonize one ormore of activin A, activin A/B, activin B, activin C, activin E, Nodal,GDF8, GDF11, BMP6 and/or BMP7. In certain embodiments, GDF traps of thepresent disclosure have diminished binding affinity for activins (e.g.,activin A, activin A/B, activin B, activin C, activin E), e.g., incomparision to a wild-type ActRII polypeptide. In certain preferredembodiments, a GDF trap polypeptide of the present disclosure hasdiminished binding affinity for activin A.

For example, the disclosure provides GDF trap polypeptides thatpreferentially bind to and/or antagonize GDF8/GDF11 relative to activinA. As demonstrated by the Examples of the disclosure, such GDF trappolypeptides are more potent activators of erythropoiesis in vivo incomparision to ActRII polypeptides that retain high binding affinity foractivin A. Furthermore, these non-activin-A-binding GDF trappolypeptides demonstrate decreased effects on other tissues. Therefore,such GDF traps may be useful for increasing red blood cell levels in asubject while reducing potential off-target effects associated withbinding/antagonizing activin A. However, such selective GDF trappolypeptides may be less desirable in some applications wherein moremodest gains in red blood cell levels may be needed for therapeuticeffect and wherein some level of off-target effect is acceptable (oreven desirable).

Amino acid residues of the ActRIIB proteins (e.g., E39, K55, Y60, K74,W78, L79, D80, and F101) are in the ActRIIB ligand-binding pocket andhelp mediated binding to its ligands including, for example, activin A,GDF11, and GDF8. Thus the present disclosure provides GDF trappolypeptides comprising an altered-ligand binding domain (e.g., aGDF8/GDF11-binding domain) of an ActRIIB receptor which comprises one ormore mutations at those amino acid residues.

Optionally, the altered ligand-binding domain can have increasedselectivity for a ligand such as GDF11 and/or GDF8 relative to awild-type ligand-binding domain of an ActRIIB receptor. To illustrate,one or more mutations may be selected that increase the selectivity ofthe altered ligand-binding domain for GDF11 and/or GDF8 over one or moreactivins (activin A, activin B, activin AB, activin C, and/or activinA), particularly activin A. Optionally, the altered ligand-bindingdomain has a ratio of K_(d) for activin binding to K_(d) for GDF11and/or GDF8 binding that is at least 2-, 5-, 10-, 20-, 50-, 100- or even1000-fold greater relative to the ratio for the wild-type ligand-bindingdomain. Optionally, the altered ligand-binding domain has a ratio ofIC₅₀ for inhibiting activin to IC₅₀ for inhibiting GDF11 and/or GDF8that is at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-fold greaterrelative to the wild-type ligand-binding domain. Optionally, the alteredligand-binding domain inhibits GDF11 and/or GDF8 with an IC₅₀ at least2-, 5-, 10-, 20-, 50-, 100- or even 1000-times less than the IC₅₀ forinhibiting activin.

As a specific example, the positively-charged amino acid residue Asp(D80) of the ligand-binding domain of ActRIIB can be mutated to adifferent amino acid residue to produce a GDF trap polypeptide thatpreferentially binds to GDF8, but not activin. Preferably, the D80residue with respect to SEQ ID NO:1 is changed to an amino acid residueselected from the group consisting of: an uncharged amino acid residue,a negative amino acid residue, and a hydrophobic amino acid residue. Asa further specific example, the hydrophobic residue L79 of SEQ ID NO:1can be altered to confer altered activin-GDF11/GDF8 binding properties.For example, an L79P substitution reduces GDF11 binding to a greaterextent than activin binding. In contrast, replacement of L79 with anacidic amino acid [an aspartic acid or glutamic acid; an L79D or an L79Esubstitution] greatly reduces activin A binding affinity while retainingGDF11 binding affinity. In exemplary embodiments, the methods describedherein utilize a GDF trap polypeptide which is a variant ActRIIBpolypeptide comprising an acidic amino acid (e.g., D or E) at theposition corresponding to position 79 of SEQ ID NO: 1, optionally incombination with one or more additional amino acid substitutions,additions, or deletions.

As will be recognized by one of skill in the art, most of the describedmutations, variants or modifications described herein may be made at thenucleic acid level or, in some cases, by post-translational modificationor chemical synthesis. Such techniques are well known in the art andsome of which are described herein.

In certain embodiments, the present disclosure relates to ActRIIpolypeptides (ActRIIA and ActRIIB polypeptides) which are soluble ActRIIpolypeptides. As described herein, the term “soluble ActRII polypeptide”generally refers to polypeptides comprising an extracellular domain ofan ActRII protein. The term “soluble ActRII polypeptide,” as usedherein, includes any naturally occurring extracellular domain of anActRII protein as well as any variants thereof (including mutants,fragments, and peptidomimetic forms) that retain a useful activity(e.g., a GDF trap polypeptide as described herein). Other examples ofsoluble ActRII polypeptides comprise a signal sequence in addition tothe extracellular domain of an ActRII or GDF trap protein. For example,the signal sequence can be a native signal sequence of an ActRIIA orActRIIB protein, or a signal sequence from another protein including,for example, a tissue plasminogen activator (TPA) signal sequence or ahoney bee melittin (HBM) signal sequence.

In part, the present disclosure identifies functionally active portionsand variants of ActRII polypeptides that can be used as guidance forgenerating and using ActRIIA polypeptides, ActRIIB polypeptides, and GDFtrap polypeptides within the scope of the methods described herein.

ActRII proteins have been characterized in the art in terms ofstructural and functional characteristics, particularly with respect toligand binding [see, e.g., Attisano et al. (1992) Cell 68(1):97-108;Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22;Allendorph et al. (2006) PNAS 103(20: 7643-7648; Thompson et al. (2003)The EMBO Journal 22(7): 1555-1566; and U.S. Pat. Nos. 7,709,605,7,612,041, and 7,842,663].

For example, Attisano et al. showed that a deletion of the proline knotat the C-terminus of the extracellular domain of ActRIIB reduced theaffinity of the receptor for activin. An ActRIIB-Fc fusion proteincontaining amino acids 20-119 of present SEQ ID NO:1,“ActRIIB(20-119)-Fc”, has reduced binding to GDF-11 and activin relativeto an ActRIIB(20-134)-Fc, which includes the proline knot region and thecomplete juxtamembrane domain (see, e.g., U.S. Pat. No. 7,842,663).However, an ActRIIB(20-129)-Fc protein retains similar but somewhatreduced activity relative to the wild-type, even though the proline knotregion is disrupted. Thus, ActRIIB extracellular domains that stop atamino acid 134, 133, 132, 131, 130 and 129 (with respect to present SEQID NO:1) are all expected to be active, but constructs stopping at 134or 133 may be most active. Similarly, mutations at any of residues129-134 (with respect to SEQ ID NO:1) are not expected to alterligand-binding affinity by large margins. In support of this, mutationsof P129 and P130 (with respect to SEQ ID NO:1) do not substantiallydecrease ligand binding. Therefore, an ActRIIB polypeptide or anActRIIB-based GDF trap polypeptide of the present disclosure may end asearly as amino acid 109 (the final cysteine), however, forms ending ator between 109 and 119 (e.g., 109, 110, 111, 112, 113, 114, 115, 116,117, 118, or 119) are expected to have reduced ligand binding. Aminoacid 119 (with respect to present SEQ ID NO:1) is poorly conserved andso is readily altered or truncated. ActRIIB polypeptides andActRIIB-based GDF traps ending at 128 (with respect to present SEQ IDNO:1) or later should retain ligand binding activity. ActRIIBpolypeptides and ActRIIB-based GDF traps ending at or between 119 and127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, or 127), with respectto SEQ ID NO:1, will have an intermediate binding ability. Any of theseforms may be desirable to use, depending on the clinical or experimentalsetting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before (with respect to present SEQ ID NO:1) willretain ligand-binding activity. Amino acid 29 represents the initialcysteine. An alanine-to-asparagine mutation at position 24 (with respectto present SEQ ID NO:1) introduces an N-linked glycosylation sequencewithout substantially affecting ligand binding (see, e.g., U.S. Pat. No.7,842,663). This confirms that mutations in the region between thesignal cleavage peptide and the cysteine cross-linked region,corresponding to amino acids 20-29, are well tolerated. In particular,ActRIIB polypeptides and ActRIIB-based GDF traps beginning at position20, 21, 22, 23, and 24 (with respect to present SEQ ID NO:1) shouldretain general ligand-biding activity, and ActRIIB polypeptides andActRIIB-based GDF traps beginning at positions 25, 26, 27, 28, and 29(with respect to present SEQ ID NO:1) are also expected to retainligand-biding activity. Data shown herein as well as in, e.g., U.S. Pat.No. 7,842,663 demonstrates that, surprisingly, an ActRIIB constructbeginning at 22, 23, 24, or 25 will have the most activity.

Taken together, an active portion (e.g., ligand-binding activity) ofActRIIB comprises amino acids 29-109 of SEQ ID NO:1. Therefore ActRIIBpolypeptides and ActRIIB-based GDF traps of the present disclosure may,for example, comprise an amino acid sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIBbeginning at a residue corresponding to amino acids 20-29 (e.g.,beginning at amino acid 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) ofSEQ ID NO: 1 and ending at a position corresponding to amino acids109-134 (e.g., ending at amino acid 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, or 134) of SEQ ID NO: 1. In some embodiments,ActRIIB-based GDF trap polypeptides of the present disclosure do notcomprise or consist of amino acids 29-109 of SEQ ID NO:1. Other examplesinclude polypeptides that begin at a position from 20-29 (e.g., position20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) or 21-29 (e.g., position 21,22, 23, 24, 25, 26, 27, 28, or 29) and end at a position from 119-134(e.g., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, or 134), 119-133 (e.g., 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, or 133), 129-134 (e.g., 129, 130,131, 132, 133, or 134), or 129-133 (e.g., 129, 130, 131, 132, or 133) ofSEQ ID NO: 1. Other examples include constructs that begin at a positionfrom 20-24 (e.g., 20, 21, 22, 23, or 24), 21-24 (e.g., 21, 22, 23, or24), or 22-25 (e.g., 22, 22, 23, or 25) and end at a position from109-134 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or134), 119-134 (e.g., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, or 134) or 129-134 (e.g., 129, 130, 131, 132,133, or 134) of SEQ ID NO: 1. Variants within these ranges are alsocontemplated, particularly those having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identity to the corresponding portion of SEQID NO: 1. In some embodiments, the ActRIIB polypeptides andActRIIB-based GDF traps comprise a polypeptide having an amino acidsequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acid residues 25-131 of SEQ ID NO: 1. In certainembodiments, ActRIIB-based GDF trap polypeptides do not comprise orconsist of amino acids 25-131 of SEQ ID NO: 1.

The disclosure includes the results of an analysis of composite ActRIIBstructures, shown in FIG. 1, demonstrating that the ligand-bindingpocket is defined, in part, by residues Y31, N33, N35, L38 through T41,E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83,Y85, R87, A92, and E94 through F101. At these positions, it is expectedthat conservative mutations will be tolerated, although a K74A mutationis well-tolerated, as are R40A, K55A, F82A and mutations at positionL79. R40 is a K in Xenopus, indicating that basic amino acids at thisposition will be tolerated. Q53 is R in bovine ActRIIB and K in XenopusActRIIB, and therefore amino acids including R, K, Q, N and H will betolerated at this position. Thus, a general formula for an ActRIIBpolypeptide and ActRIIB-based GDF trap polypeptide of the disclosure isone that comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 ofSEQ ID NO: 1, optionally beginning at a position ranging from 20-24(e.g., 20, 21, 22, 23, or 24) or 22-25 (e.g., 22, 23, 24, or 25) andending at a position ranging from 129-134 (e.g., 129, 130, 131, 132,133, or 134), and comprising no more than 1, 2, 5, 10 or 15 conservativeamino acid changes in the ligand-binding pocket, and zero, one or morenon-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82in the ligand-binding pocket. Sites outside the binding pocket, at whichvariability may be particularly well tolerated, include the amino andcarboxy termini of the extracellular domain (as noted above), andpositions 42-46 and 65-73 (with respect to SEQ ID NO:1). Anasparagine-to-alanine alteration at position 65 (N65A) actually improvesligand binding in the A64 background, and is thus expected to have nodetrimental effect on ligand binding in the R64 background (see, e.g.,U.S. Pat. No. 7,842,663). This change probably eliminates glycosylationat N65 in the A64 background, thus demonstrating that a significantchange in this region is likely to be tolerated. While an R64A change ispoorly tolerated, R64K is well-tolerated, and thus another basicresidue, such as H may be tolerated at position 64 (see, e.g., U.S. Pat.No. 7,842,663).

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered. Therefore, anactive, human ActRIIB variant polypeptide and ActRIIB-based GDF trapuseful in accordance with the presently disclosed methods may includeone or more amino acids at corresponding positions from the sequence ofanother vertebrate ActRIIB, or may include a residue that is similar tothat in the human or other vertebrate sequence. The following examplesillustrate this approach to defining an active ActRIIB variant. L46 is avaline in Xenopus ActRIIB, and so this position may be altered, andoptionally may be altered to another hydrophobic residue, such as V, Ior F, or a non-polar residue such as A. E52 is a K in Xenopus,indicating that this site may be tolerant of a wide variety of changes,including polar residues, such as E, D, K, R, H, S, T, P, G, Y andprobably A. T93 is a K in Xenopus, indicating that a wide structuralvariation is tolerated at this position, with polar residues favored,such as S, K, R, E, D, H, G, P, G and Y. F108 is a Yin Xenopus, andtherefore Y or other hydrophobic group, such as I, V or L should betolerated. E111 is K in Xenopus, indicating that charged residues willbe tolerated at this position, including D, R, K and H, as well as Q andN. R112 is K in Xenopus, indicating that basic residues are tolerated atthis position, including R and H. A at position 119 is relatively poorlyconserved, and appears as P in rodents and V in Xenopus, thusessentially any amino acid should be tolerated at this position.

It has been previously demonstrated that the addition of a furtherN-linked glycosylation site (N-X-S/T) is well-tolerated relative to theActRIIB(R64)-Fc form (see, e.g., U.S. Pat. No. 7,842,663). Therefore,N-X-S/T sequences may be generally introduced at positions outside theligand binding pocket defined in FIG. 1 in ActRIIB polypeptide andActRIIB-based GDF traps of the present disclosure. Particularly suitablesites for the introduction of non-endogenous N-X-S/T sequences includeamino acids 20-29, 20-24, 22-25, 109-134, 120-134 or 129-134 (withrespect to SEQ ID NO:1). N-X-S/T sequences may also be introduced intothe linker between the ActRIIB sequence and an Fc domain or other fusioncomponent. Such a site may be introduced with minimal effort byintroducing an N in the correct position with respect to a pre-existingS or T, or by introducing an S or T at a position corresponding to apre-existing N. Thus, desirable alterations that would create anN-linked glycosylation site are: A24N, R64N, S67N (possibly combinedwith an N65A alteration), E105N, R112N, G120N, E123N, P129N, A132N,R112S and R112T (with respect to SEQ ID NO:1). Any S that is predictedto be glycosylated may be altered to a T without creating an immunogenicsite, because of the protection afforded by the glycosylation. Likewise,any T that is predicted to be glycosylated may be altered to an S. Thusthe alterations S67T and S44T (with respect to SEQ ID NO:1) arecontemplated. Likewise, in an A24N variant, an S26T alteration may beused. Accordingly, an ActRIIB polypeptide and ActRIIB-based GDF trappolypeptide of the present disclosure may be a variant having one ormore additional, non-endogenous N-linked glycosylation consensussequences as described above.

The variations described herein may be combined in various ways.Additionally, the results of the mutagenesis program described hereinindicate that there are amino acid positions in ActRIIB that are oftenbeneficial to conserve. With respect to SEQ ID NO:1, these includeposition 64 (basic amino acid), position 80 (acidic or hydrophobic aminoacid), position 78 (hydrophobic, and particularly tryptophan), position37 (acidic, and particularly aspartic or glutamic acid), position 56(basic amino acid), position 60 (hydrophobic amino acid, particularlyphenylalanine or tyrosine). Thus, in the ActRIIB polypeptides andActRIIB-based GDF traps disclosed herein, the disclosure provides aframework of amino acids that may be conserved. Other positions that maybe desirable to conserve are as follows: position 52 (acidic aminoacid), position 55 (basic amino acid), position 81 (acidic), 98 (polaror charged, particularly E, D, R or K), all with respect to SEQ ID NO:1.

A general formula for an active (e.g., ligand binding) ActRIIApolypeptide is one that comprises a polypeptide that starts at aminoacid 30 and ends at amino acid 110 of SEQ ID NO:9. Accordingly, ActRIIApolypeptides and ActRIIA-based GDF traps of the present disclosure maycomprise a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO:9. Insome embodiments, ActRIIA-based GDF traps of the present disclosure donot comprise or consist of amino acids 30-110 of SEQ ID NO:9.Optionally, ActRIIA polypeptides and ActRIIA-based GDF trap polypeptidesof the present disclosure comprise a polypeptide that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acidsamino acids 12-82 of SEQ ID NO:9 optionally beginning at a positionranging from 1-5 (e.g., 1, 2, 3, 4, or 5) or 3-5 (e.g., 3, 4, or 5) andending at a position ranging from 110-116 (e.g., 110, 111, 112, 113,114, 115, or 116) or 110-115 (e.g., 110, 111, 112, 113, 114, or 115),respectively, and comprising no more than 1, 2, 5, 10 or 15 conservativeamino acid changes in the ligand binding pocket, and zero, one or morenon-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82in the ligand-binding pocket with respect to SEQ ID NO:9.

In certain embodiments, functionally active fragments of ActRIIpolypeptides (e.g. ActRIIA and ActRIIB polypeptides) and GDF trappolypeptides of the present disclosure can be obtained by screeningpolypeptides recombinantly produced from the corresponding fragment ofthe nucleic acid encoding an ActRII polypeptide or GDF trap polypeptide(e.g., SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 46, and 48). Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments that canfunction as antagonists (inhibitors) of ActRII receptors and/or one ormore ActRII ligands (e.g., GDF11, GDF8, activin A, activin B, activinAB, activin C, activin E, BMP6, BMP7, and/or Nodal).

In some embodiments, an ActRIIA polypeptide of the present disclosure isa polypeptide comprising an amino acid sequence that is at least 75%identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11,22, 26, and 28. In certain embodiments, the ActRIIA polypeptidecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to an amino acid sequence selected fromSEQ ID NOs: 9, 10, 11, 22, 26, and 28. In certain embodiments, theActRIIA polypeptide consists essentially of, or consists of, an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11,22, 26, and 28.

In some embodiments, an ActRIIB polypeptide of the present disclosure isa polypeptide comprising an amino acid sequence that is at least 75%identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3,4, 5, 6, 29, 31, and 49. In certain embodiments, the ActRIIB polypeptidecomprises an amino acid sequence that is at least 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to an amino acid sequence selected fromSEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 31, and 49. In certain embodiments,the ActRIIB polypeptide consists essentially of, or consists of, anamino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1,2, 3, 4, 5, 6, 29, 31, and 49.

In some embodiments, a GDF trap polypeptide of the present disclosure isa variant ActRIIB polypeptide comprising an amino acid sequence that isat least 75% identical to an amino acid sequence selected from SEQ IDNOs: 1, 2, 3, 4, 5, 6, 29, 30, 31, 36, 37, 38, 41, 44, 45, 49, 50, and51. In certain embodiments, the GDF trap comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3,4, 5, 6, 29, 30, 31, 36, 37, 38, 41, 44, 45, 49, 50, and 51. In certainembodiments, the GDF trap comprises an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 30, 31,36, 37, 38, 41, 44, 45, 49, 50, and 51, wherein the positioncorresponding to L79 of SEQ ID NO:1, 4, or 49 is an acidic amino acids(a D or E amino acid residue). In certain embodiments, the GDF trapconsists essentially of, or consists of, an amino acid sequence that atleast 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from SEQ ID NOs: 36, 37, 38, 41, 44, 45, 50, and51. In certain embodiments, the GDF trap does not comprise or consistsof an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6,29, and 31.

In some embodiments, a GDF trap polypeptide of the present disclosure isa variant ActRIIA polypeptide comprising an amino acid sequence that isat least 75% identical to an amino acid sequence selected from SEQ IDNOs: 9, 10, 11, 22, 26, 28, 29, and 31. In certain embodiments, the GDFtrap comprises an amino acid sequence that is at least 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selectedfrom SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31. In certainembodiments, the GDF trap consists essentially of, or consists of, anamino acid sequence that at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or100% identical to an amino acid sequence selected from SEQ ID NOs: 9,10, 11, 22, 26, 28, 29, and 31. In certain embodiments, the GDF trapdoes not comprise or consists of an amino acid sequence selected fromSEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31.

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of an ActRII polypeptide(e.g. and ActRIIA or ActRIIB polypeptide) or a GDF trap for suchpurposes as enhancing therapeutic efficacy, or stability (e.g.,shelf-life and resistance to proteolytic degradation in vivo). Variantscan be produced by amino acid substitution, deletion, addition, orcombinations thereof. For instance, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Whether a change in the amino acidsequence of a polypeptide of the disclosure results in a functionalhomolog can be readily determined by assessing the ability of thevariant polypeptide to produce a response in cells in a fashion similarto the wild-type polypeptide, or to bind to one or more ligands, such asGDF11, activin A, activin B, activin AB, activin C, activin E, GDF8,BMP6, and BMP7, as compared to the unmodified or a wild-typepolypeptide.

In certain embodiments, the present disclosure contemplates specificmutations of ActRII polypeptides and GDF trap polypeptides of thepresent disclosure so as to alter the glycosylation of the polypeptide.Such mutations may be selected so as to introduce or eliminate one ormore glycosylation sites, such as O-linked or N-linked glycosylationsites. Asparagine-linked glycosylation recognition sites generallycomprise a tripeptide sequence, asparagine-X-threonine orasparagine-X-serine (where “X” is any amino acid) which is specificallyrecognized by appropriate cellular glycosylation enzymes. The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the polypeptide (forO-linked glycosylation sites). A variety of amino acid substitutions ordeletions at one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal ofone or more carbohydrate moieties present on a polypeptide may beaccomplished chemically and/or enzymatically. Chemical deglycosylationmay involve, for example, exposure of a polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987)138:350]. The sequence of a polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect, and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, ActRII polypeptides and GDF trap polypeptides of the presentdisclosure for use in humans may be expressed in a mammalian cell linethat provides proper glycosylation, such as HEK293 or CHO cell lines,although other mammalian expression cell lines are expected to be usefulas well.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of ActRII polypeptides andGDF trap polypeptides of the present disclosure, as well as truncationmutants. Pools of combinatorial mutants are especially useful foridentifying ActRII and GDF trap sequences. The purpose of screening suchcombinatorial libraries may be to generate, for example, polypeptidesvariants which have altered properties, such as altered pharmacokineticor altered ligand binding. A variety of screening assays are providedbelow, and such assays may be used to evaluate variants. For example,ActRII polypeptides and GDF trap polypeptides may be screened forability to bind to an ActRII receptor, to prevent binding of an ActRIIligand (e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C,activin E, BMP7, BMP6, and/or Nodal) to an ActRII polypeptide, or tointerfere with signaling caused by an ActRII ligand.

The activity of an ActRII polypeptides or GDF trap polypeptides may alsobe tested in a cell-based or in vivo assay. For example, the effect ofan ActRII polypeptide or GDF trap polypeptide on the expression of genesinvolved in hematopoiesis may be assessed. This may, as needed, beperformed in the presence of one or more recombinant ActRII ligandproteins (e.g., GDF11, GDF8, activin A, activin B, activin AB, activinC, activin E, BMP7, BMP6, and/or Nodal), and cells may be transfected soas to produce an ActRII polypeptide or GDF trap polypeptide, andoptionally, an ActRII ligand. Likewise, an ActRII polypeptide or GDFtrap polypeptide may be administered to a mouse or other animal, and oneor more blood measurements, such as an RBC count, hemoglobin, orreticulocyte count may be assessed using art-recognized methods.

Combinatorial-derived variants can be generated which have a selectiveor generally increased potency relative to a reference ActRIIpolypeptide or GDF trap polypeptide. Such variants, when expressed fromrecombinant DNA constructs, can be used in gene therapy protocols.Likewise, mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding unmodifiedActRII polypeptide or GDF trap polypeptide. For example, the alteredprotein can be rendered either more stable or less stable to proteolyticdegradation or other cellular processes which result in destruction, orotherwise inactivation, of an unmodified polypeptide. Such variants, andthe genes which encode them, can be utilized to alter ActRII polypeptideor GDF trap polypeptide levels by modulating the half-life of thepolypeptide. For instance, a short half-life can give rise to moretransient biological effects and, when part of an inducible expressionsystem, can allow tighter control of recombinant ActRII polypeptide orGDF trap polypeptide levels within the cell. In an Fc fusion protein,mutations may be made in the linker (if any) and/or the Fc portion toalter the half-life of the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ActRII or GDF trap sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ActRII or GDFtrap polypeptide encoding nucleotide sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art. See, e.g., Narang, S A (1983)Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rdCleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins. See, e.g., Scott et al., (1990) Science 249:386-390; Robertset al. (1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249:404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815.

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ActRII polypeptides or GDF trappolypeptides of the present disclosure can be generated and isolatedfrom a library by screening using, for example, alanine scanningmutagenesis [see, e.g., Ruf et al. (1994) Biochemistry 33:1565-1572;Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint et al. (1993)Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601;Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.(1991) Biochemistry 30:10832-10838; and Cunningham et al. (1989) Science244:1081-1085], by linker scanning mutagenesis [see, e.g., Gustin et al.(1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol.12:2644-2652; McKnight et al. (1982) Science 232:316], by saturationmutagenesis [see, e.g., Meyers et al., (1986) Science 232:613]; by PCRmutagenesis [see, e.g., Leung et al. (1989) Method Cell Mol Biol1:11-19]; or by random mutagenesis, including chemical mutagenesis [see,e.g., Miller et al. (1992) A Short Course in Bacterial Genetics, CSHLPress, Cold Spring Harbor, N.Y.; and Greener et al. (1994) Strategies inMol Biol 7:32-34]. Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ActRII polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ActRII polypeptides or GDF trappolypeptides of the disclosure. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Preferred assays include ActRIIligand (e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C,activin E, BMP7, BMP6, and/or Nodal) binding assays and/or ActRIIligand-mediated cell signaling assays.

In certain embodiments, ActRII polypeptides or GDF trap polypeptides ofthe present disclosure may further comprise post-translationalmodifications in addition to any that are naturally present in theActRII (e.g. an ActRIIA or ActRIIB polypeptide) or GDF trap polypeptide.Such modifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the ActRII polypeptide or GDF trap polypeptidemay contain non-amino acid elements, such as polyethylene glycols,lipids, polysaccharide or monosaccharide, and phosphates. Effects ofsuch non-amino acid elements on the functionality of a ligand trappolypeptide may be tested as described herein for other ActRII or GDFtrap variants. When a polypeptide of the disclosure is produced in cellsby cleaving a nascent form of the polypeptide, post-translationalprocessing may also be important for correct folding and/or function ofthe protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3or HEK293) have specific cellular machinery and characteristicmechanisms for such post-translational activities and may be chosen toensure the correct modification and processing of the ActRIIpolypeptides or GDF trap polypeptides.

In certain aspects, ActRII polypeptides or GDF trap polypeptides of thepresent disclosure include fusion proteins having at least a portion(domain) of an ActRII polypeptide (e.g., an ActRIIA or ActRIIBpolypeptide) or GDF trap polypeptide and one or more heterologousportions (domains). Well-known examples of such fusion domains include,but are not limited to, polyhistidine, Glu-Glu, glutathioneS-transferase (GST), thioredoxin, protein A, protein G, animmunoglobulin heavy-chain constant region (Fc), maltose binding protein(MBP), or human serum albumin. A fusion domain may be selected so as toconfer a desired property. For example, some fusion domains areparticularly useful for isolation of the fusion proteins by affinitychromatography. For the purpose of affinity purification, relevantmatrices for affinity chromatography, such as glutathione-, amylase-,and nickel- or cobalt-conjugated resins are used. Many of such matricesare available in “kit” form, such as the Pharmacia GST purificationsystem and the QIAexpress' system (Qiagen) useful with (HIS6) fusionpartners. As another example, a fusion domain may be selected so as tofacilitate detection of the ligand trap polypeptides. Examples of suchdetection domains include the various fluorescent proteins (e.g., GFP)as well as “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well-known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orthrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation. In certain preferred embodiments,an ActRII polypeptide or a GDF trap polypeptide is fused with a domainthat stabilizes the polypeptide in vivo (a “stabilizer” domain). By“stabilizing” is meant anything that increases serum half-life,regardless of whether this is because of decreased destruction,decreased clearance by the kidney, or other pharmacokinetic effect.Fusions with the Fc portion of an immunoglobulin are known to conferdesirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherstimulation of muscle growth).

In certain embodiments, the present disclosure provides ActRII or GDFtrap fusion proteins comprising the following IgG1 Fc domain sequence:

(SEQ ID NO: 14) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPVPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK.

In other embodiments, the present disclosure provides ActRII or GDF trapfusion proteins comprising the following variant of the IgG1 Fc domain:

(SEQ ID NO: 64) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK

In still other embodiments, the present disclosure provides ActRII orGDF trap fusion proteins comprising the following variant of the IgG1 Fcdomain:

(SEQ ID NO: 15) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCVVVD(A)VSHEDPE 51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK(A) 101 VSNKALPVPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG PFFLYSKLTV DKSRWQQGNV 201FSCSVMHEAL HN(A)HYTQKSLS LSPGK.

Optionally, the IgG1 Fc domain has one or more mutations at residuessuch as Asp-265, lysine 322, and Asn-434. In certain cases, the mutantIgG1 Fc domain having one or more of these mutations (e.g., Asp-265mutation) has reduced ability of binding to the Fey receptor relative toa wild-type Fc domain. In other cases, the mutant Fc domain having oneor more of these mutations (e.g., Asn-434 mutation) has increasedability of binding to the MHC class I-related Fc-receptor (FeRN)relative to a wild-type IgG1 Fc domain.

In certain other embodiments, the present disclosure provides ActRII orGDF trap fusion proteins comprising variants of the IgG2 Fc domain,including the following:

(SEQ ID NO: 65) 1 VECPPCPAPP VAGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVQ51 FNWYVDGVEV HNAKTKPREE QFNSTFRVVS VLTVVHQDWL NGKEYKCKVS 101 NKGLPAPIEKTISKTKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP 151 SDIAVEWESN GQPENNYKTTPPMLDSDGSF FLYSKLTVDK SRWQQGNVFS 201 CSVMHEALHN HYTQKSLSLS PGK

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRII polypeptide domain or GDF trappolypeptide domain may be placed C-terminal to a heterologous domain, oralternatively, a heterologous domain may be placed C-terminal to anActRII polypeptide domain or GDF trap polypeptide domain. The ActRIIpolypeptide domain or GDF trap polypeptide domain and the heterologousdomain need not be adjacent in a fusion protein, and additional domainsor amino acid sequences may be included C- or N-terminal to eitherdomain or between the domains.

For example, an ActRII or GDF trap fusion protein may comprise an aminoacid sequence as set forth in the formula A-B-C. The B portioncorresponds to an ActRII polypeptide domain or a GDF trap polypeptidedomain. The A and C portions may be independently zero, one, or morethan one amino acid, and both the A and C portions when present areheterologous to B. The A and/or C portions may be attached to the Bportion via a linker sequence. Exemplary linkers include shortpolypeptide linkers such as 2-10, 2-5, 2-4, 2-3 glycine residues, suchas, for example, a Gly-Gly-Gly linker. Other suitable linkers aredescribed herein above [e.g., a TGGG linker (SEQ ID NO: 53)]. In certainembodiments, an ActRII or GDF trap fusion protein comprises an aminoacid sequence as set forth in the formula A-B-C, wherein A is a leader(signal) sequence, B consists of an ActRII or GDF polypeptide domain,and C is a polypeptide portion that enhances one or more of in vivostability, in vivo half-life, uptake/administration, tissue localizationor distribution, formation of protein complexes, and/or purification. Incertain embodiments, an ActRII or GDF trap fusion protein comprises anamino acid sequence as set forth in the formula A-B-C, wherein A is aTPA leader sequence, B consists of an ActRII or GDF polypeptide domain,and C is an immunoglobulin Fc domain. Preferred fusion proteinscomprises the amino acid sequence set forth in any one of SEQ ID NOs:22, 26, 29, 31, 36, 38, 41, 44, and 51.

In certain embodiments, ActRII polypeptides or GDF trap polypeptides ofthe present disclosure contain one or more modifications that arecapable of stabilizing the polypeptides. For example, such modificationsenhance the in vitro half-life of the polypeptides, enhance circulatoryhalf-life of the polypeptides, and/or reduce proteolytic degradation ofthe polypeptides. Such stabilizing modifications include, but are notlimited to, fusion proteins (including, for example, fusion proteinscomprising an ActRII polypeptide domain or a GDF trap polypeptide domainand a stabilizer domain), modifications of a glycosylation site(including, for example, addition of a glycosylation site to apolypeptide of the disclosure), and modifications of carbohydrate moiety(including, for example, removal of carbohydrate moieties from apolypeptide of the disclosure). As used herein, the term “stabilizerdomain” not only refers to a fusion domain (e.g., an immunoglobulin Fcdomain) as in the case of fusion proteins, but also includesnonproteinaceous modifications such as a carbohydrate moiety, ornonproteinaceous moiety, such as polyethylene glycol.

In preferred embodiments, ActRII polypeptides and GDF traps to be usedin accordance with the methods described herein are isolatedpolypeptides. As used herein, an isolated protein or polypeptide is onewhich has been separated from a component of its natural environment. Insome embodiments, a polypeptide of the disclosure is purified to greaterthan 95%, 96%, 97%, 98%, or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). Methods for assessment of antibody purity are well known in theart [see, e.g., Flatman et al., (2007) J. Chromatogr. B 848:79-87].

In certain embodiments, ActRII polypeptides and GDF traps of thedisclosure can be produced by a variety of art-known techniques. Forexample, polypeptides of the disclosure can be synthesized usingstandard protein chemistry techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). In addition, automated peptidesynthesizers are commercially available (see, e.g., Advanced ChemTechModel 396; Milligen/Biosearch 9600). Alternatively, the polypeptides ofthe disclosure, including fragments or variants thereof, may berecombinantly produced using various expression systems [e.g., E. coli,Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus] as is wellknown in the art. In a further embodiment, the modified or unmodifiedpolypeptides of the disclosure may be produced by digestion ofrecombinantly produced full-length ActRII or GDF trap polypeptides byusing, for example, a protease, e.g., trypsin, thermolysin,chymotrypsin, pepsin, or paired basic amino acid converting enzyme(PACE). Computer analysis (using a commercially available software,e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be usedto identify proteolytic cleavage sites. Alternatively, such polypeptidesmay be produced from recombinantly produced full-length ActRII or GDFtrap polypeptides using chemical cleavage (e.g., cyanogen bromide,hydroxylamine, etc.).

Any of the ActRII polypeptides disclosed herein (e.g., ActRIIA orActRIIB polypeptides) can be combined with one or more additional ActRIIantagonist agents of the disclosure to achieve the desired effect (e.g.,increase red blood cell levels and/or hemoglobin in a subject in needthereof, treat or prevent an anemia, treat MDS or sideroblastic anemias,treat or prevent one or more complications of MDS or sideroblasticanemias s). For example, an ActRII polypeptide disclosed herein can beused in combination with i) one or more additional ActRII polypeptidesdisclosed herein, ii) one or more GDF traps disclosed herein; iii) oneor more ActRII antagonist antibodies disclosed herein (e.g., ananti-activin A antibody, an anti-activin B antibody, an anti-activin Cantibody, an anti-activin E antibody, an anti-GDF11 antibody, ananti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, ananti-ActRIIA antibody, and/or or an anti-ActRIIB antibody); iv) one ormore small-molecule ActRII antagonists disclosed herein (e.g., asmall-molecule antagonist of one or more of GDF11, GDF8, activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and/or ActRIIB); v) one or more of the polynucleotide ActRII antagonistsdisclosed herein (e.g., a polynucleotide antagonist of one or more ofGDF11, GDF8, activin A, activin B, activin AB, activin C, activin E,BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); vi) one or more follistatinpolypeptides disclosed herein; and/or vii) one or more FLRG polypeptidesdisclosed herein.

Similarly, any of the GDF traps disclosed herein can be combined withone or more additional ActRII antagonist agents of the disclosure toachieve the desired effect (e.g., increase red blood cell levels and/orhemoglobin in a patient in need thereof, treat or prevent an anemia,treat MDS or sideroblastic anemias, treat or prevent one or morecomplications of MDS or sideroblastic anemias). For example, a GDF trapdisclosed herein can be used in combination with i) one or moreadditional GDF traps disclosed herein, ii) one or more ActRIIpolypeptides disclosed herein (e.g., ActRIIA or ActRIIB polypeptides)disclosed herein; iii) one or more ActRII antagonist antibodiesdisclosed herein (e.g., an anti-activin A antibody, an anti-activin Bantibody, an anti-activin C antibody, an anti-activin E antibody, ananti-GDF11 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, ananti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an anti-ActRIIBantibody); iv) one or more small-molecule ActRII antagonists disclosedherein (e.g., a small-molecule antagonist of one or more of GDF11, GDF8,activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7,Nodal, ActRIIA, and/or ActRIIB); v) one or more of the polynucleotideActRII antagonists disclosed herein (e.g., a polynucleotide antagonistof one or more of GDF11, GDF8, activin A, activin B, activin AB, activinC, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); vi) one ormore follistatin polypeptides disclosed herein; and/or vii) one or moreFLRG polypeptides disclosed herein.

B. Nucleic Acids Encoding ActRII Polypeptides and GDF Traps

In certain embodiments, the present disclosure provides isolated and/orrecombinant nucleic acids encoding the ActRII polypeptides and GDF trappolypeptides (including fragments, functional variants, and fusionproteins thereof) disclosed herein. For example, SEQ ID NO:12 encodesthe naturally occurring human ActRIIA precursor polypeptide, while SEQID NO:13 encodes the processed extracellular domain of ActRIIA. Inaddition, SEQ ID NO:7 encodes a naturally occurring human ActRIIBprecursor polypeptide (the R64 variant described above), while SEQ IDNO:8 encodes the processed extracellular domain of ActRIIB (the R64variant described above). The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids may be used, for example, in methods formaking ActRII-based ligand trap polypeptides of the present disclosure.

As used herein, isolated nucleic acid(s) refers to a nucleic acidmolecule that has been separated from a component of its naturalenvironment. An isolated nucleic acid includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

In certain embodiments, nucleic acids encoding ActRII polypeptides andGDF traps of the present disclosure are understood to include nucleicacids that are variants of any one of SEQ ID NOs: 7, 8, 12, 13, 27, 32,39, 40, 42, 43, 46, 47, and 48. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions,additions, or deletions including allelic variants, and therefore, willinclude coding sequence that differ from the nucleotide sequencedesignated in any one of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42,43, 46, 47, and 48.

In certain embodiments, ActRII polypeptides and GDF traps of the presentdisclosure are encoded by isolated or recombinant nucleic acid sequencesthat are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48 In someembodiments, GDF traps of the present disclosure are not encoded bynucleic acid sequences that comprise or consist of any one of nucleotidesequences corresponding to any one of SEQ ID NOs: 7, 8, 12, 13, 27, and32. One of ordinary skill in the art will appreciate that nucleic acidsequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to the sequences complementary to SEQ ID NOs: 7, 8, 12, 13,27, 32, 39, 42, 47, and 48, and variants thereof, are also within thescope of the present disclosure. In further embodiments, the nucleicacid sequences of the disclosure can be isolated, recombinant, and/orfused with a heterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the present disclosure alsoinclude nucleotide sequences that hybridize under highly stringentconditions to the nucleotide sequence designated in SEQ ID NOs: 7, 8,12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48, complement sequences ofSEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48, orfragments thereof. As discussed above, one of ordinary skill in the artwill understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. One of ordinary skill in theart will understand readily that appropriate stringency conditions whichpromote DNA hybridization can be varied. For example, one could performthe hybridization at 6.0× sodium chloride/sodium citrate (SSC) at about45° C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48 dueto degeneracy in the genetic code are also within the scope of thedisclosure. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonyms(for example, CAU and CAC are synonyms for histidine) may result in“silent” mutations which do not affect the amino acid sequence of theprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this disclosure.

In certain embodiments, the recombinant nucleic acids of the presentdisclosure may be operably linked to one or more regulatory nucleotidesequences in an expression construct. Regulatory nucleotide sequenceswill generally be appropriate to the host cell used for expression.Numerous types of appropriate expression vectors and suitable regulatorysequences are known in the art for a variety of host cells. Typically,said one or more regulatory nucleotide sequences may include, but arenot limited to, promoter sequences, leader or signal sequences,ribosomal binding sites, transcriptional start and terminationsequences, translational start and termination sequences, and enhanceror activator sequences. Constitutive or inducible promoters as known inthe art are contemplated by the disclosure. The promoters may be eithernaturally occurring promoters, or hybrid promoters that combine elementsof more than one promoter. An expression construct may be present in acell on an episome, such as a plasmid, or the expression construct maybe inserted in a chromosome. In some embodiments, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the present disclosure, the subject nucleic acidis provided in an expression vector comprising a nucleotide sequenceencoding an ActRII polypeptide or a GDF trap and operably linked to atleast one regulatory sequence. Regulatory sequences are art-recognizedand are selected to direct expression of the ActRII or GDF trappolypeptide. Accordingly, the term regulatory sequence includespromoters, enhancers, and other expression control elements. Exemplaryregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences that control the expression of a DNA sequence when operativelylinked to it may be used in these vectors to express DNA sequencesencoding an ActRII or GDF trap polypeptide. Such useful expressioncontrol sequences, include, for example, the early and late promoters ofSV40, tet promoter, adenovirus or cytomegalovirus immediate earlypromoter, RSV promoters, the lac system, the trp system, the TAC or TRCsystem, T7 promoter whose expression is directed by T7 RNA polymerase,the major operator and promoter regions of phage lambda, the controlregions for fd coat protein, the promoter for 3-phosphoglycerate kinaseor other glycolytic enzymes, the promoters of acid phosphatase, e.g.,Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the present disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant ActRII or GDF trap polypeptide include plasmids andother vectors. For instance, suitable vectors include plasmids of thefollowing types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see, e.g., Molecular Cloning A LaboratoryManual, 3rd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press, 2001). In some instances, it may be desirableto express the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRII or GDF trap polypeptides in CHO cells, such as aPcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors(Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison,Wis.). As will be apparent, the subject gene constructs can be used tocause expression of the subject ActRII polypeptides in cells propagatedin culture, e.g., to produce proteins, including fusion proteins orvariant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject ActRII or GDF trap polypeptides. The host cell may be anyprokaryotic or eukaryotic cell. For example, an ActRII or GDF trappolypeptide of the disclosure may be expressed in bacterial cells suchas E. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells [e.g. a Chinese hamster ovary (CHO) cellline]. Other suitable host cells are known to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject ActRII and GDF trap polypeptides. For example, ahost cell transfected with an expression vector encoding an ActRII orGDF trap polypeptide can be cultured under appropriate conditions toallow expression of the ActRII or GDF trap polypeptide to occur. Thepolypeptide may be secreted and isolated from a mixture of cells andmedium containing the polypeptide. Alternatively, the ActRII or GDF trappolypeptide may be retained cytoplasmically or in a membrane fractionand the cells harvested, lysed and the protein isolated. A cell cultureincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. The subject polypeptides can beisolated from cell culture medium, host cells, or both, using techniquesknown in the art for purifying proteins, including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, immunoaffinity purification with antibodies specificfor particular epitopes of the ActRII or GDF trap polypeptides, andaffinity purification with an agent that binds to a domain fused to theActRII or GDF trap polypeptide (e.g., a protein A column may be used topurify an ActRII-Fc or GDF Trap-Fc fusion protein). In some embodiments,the ActRII or GDF trap polypeptide is a fusion protein containing adomain which facilitates its purification.

In some embodiments, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. AnActRII-Fc or GDF trap-Fc protein may be purified to a purityof >90%, >95%, >96%, >98%, or >99% as determined by size exclusionchromatography and >90%, >95%, >96%, >98%, or >99% as determined by SDSPAGE. The target level of purity should be one that is sufficient toachieve desirable results in mammalian systems, particularly non-humanprimates, rodents (mice), and humans.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRII or GDFtrap polypeptide, can allow purification of the expressed fusion proteinby affinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRII or GDF trap polypeptide.See, e.g., Hochuli et al. (1987) J. Chromatography 411:177; andJanknecht et al. (1991) PNAS USA 88:8972.

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence. See,e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., JohnWiley & Sons: 1992.

C. Antibody Antagonists

In certain aspects, the present disclosure relates to an antibody, orcombination of antibodies, that antagonize ActRII activity (e.g.,inhibition of ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 and/or SMAD 1/5/8 signaling). Such antibodies may bind to andinhibit one or more ligands (e.g., GDF8, GDF11, activin A, activin B,activin C, activin E, BMP6, BMP7 or Nodal) or one or more receptors(e.g., ActRIIA, ActRIIB, ALK4, ALK5). In particular, the disclosureprovides methods of using an antibody ActRII antagonist, or combinationof antibody ActRII antagonists, alone or in combination with one or moreerythropoiesis stimulating agents (e.g., EPO) or other supportivetherapies [e.g., hematopoietic growth factors (e.g., G-CSF or GM-CSF),transfusion of red blood cells or whole blood, iron chelation therapy],to, e.g., increase red blood cell levels in a subject in need thereof,treat or prevent an anemia in a subject in need thereof (including,e.g., reduction of transfusion burden), treat MDS or sideroblasticanemias in a subject in need thereof, and/or treat or prevent one ormore complications of MDS or sideroblastic anemias (e.g., anemia, bloodtransfusion requirement, neutropenia, iron overload, acute myocardialinfarction, hepatic failure, hepatomegaly, splenomegaly, progression toacute myeloid lymphoma) and or treat or prevent a disorder associatedwith SF3B1, DNMT3A, and/or TET2 mutations in a subject in need thereof.

In certain embodiments, a preferred antibody ActRII antagonist of thedisclosure is an antibody, or combination of antibodies, that binds toand/or inhibits activity of at least GDF11 (e.g., GDF11-mediatedactivation of ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 signaling). Optionally, the antibody, or combination ofantibodies, further binds to and/or inhibits activity of GDF8 (e.g.,GDF8-mediated activation of ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling), particularly in the case of amultispecific antibody that has binding affinity for both GDF11 and GDF8or in the context of a combination of one or more anti-GDF11 antibodiesand one or more anti-GDF8 antibodies. Optionally, an antibody, orcombination of antibodies, of the disclosure does not substantially bindto and/or inhibit activity of activin A (e.g., activin A-mediatedactivation of ActRIIA or ActRIIB signaling transduction, such as SMAD2/3 signaling). In some embodiments, an antibody, or combination ofantibodies, of the disclosure that binds to and/or inhibits the activityof GDF11 and/or GDF8 further binds to and/or inhibits activity of one ofmore of activin A, activin B, activin AB, activin C, activin E, BMP6,BMP7, and Nodal (e.g., activation of ActRIIA or ActRIIB SMAD 2/3 and/orSMAD 1/5/8 signaling), particularly in the case of a multispecificantibody that has binding affinity for multiple ActRII ligands or in thecontext of a combination of multiple antibodies—each having bindingaffinity for a different ActRII ligand.

In certain aspects, an ActRII antagonist of the present disclosure is anantibody, or combination of antibodies, that binds to and/or inhibitsactivity of at least GDF8 (e.g., GDF8-mediated activation of ActRIIAand/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).Optionally, the antibody, or combination of antibodies, further binds toand/or inhibits activity of GDF11 (e.g., GDF11-mediated activation ofActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3signaling), particularly in the case of a multispecific antibody thathas binding affinity for both GDF8 and GDF11 or in the context of acombination of one or more anti-GDF8 antibodies and one or moreanti-GDF11 antibodies. Optionally, an antibody, or combination ofantibodies, of the disclosure does not substantially bind to and/orinhibit activity of activin A (e.g., activin A-mediated activation ofActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 signaling).In some embodiments, an antibody, or combination of antibodies, of thedisclosure that binds to and/or inhibits the activity of GDF8 and/orGDF11 further binds to and/or inhibits activity of one of more ofactivin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, andNodal (e.g., activation of ActRIIA or ActRIIB signaling transduction,such as SMAD 2/3 and/or SMAD 1/5/8 signaling), particularly in the caseof a multispecific antibody that has binding affinity for multipleActRII ligands or in the context of a combination multipleantibodies—each having binding affinity for a different ActRII ligand.

In another aspect, an ActRII antagonist of the present disclosure is anantibody, or combination of antibodies, that binds to and/or inhibitsactivity of an ActRII receptor (e.g. an ActRIIA or ActRIIB receptor). Inpreferred embodiments, an anti-ActRII receptor antibody (e.g. ananti-ActRIIA or anti-ActRIIB receptor antibody), or combination ofantibodies, of the disclosure binds to an ActRII receptor and preventsbinding and/or activation of the ActRII receptor by at least GDF11(e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling). Optionally, an anti-ActRIIreceptor antibody, or combination of antibodies, of the disclosurefurther prevents binding and/or activation of the ActRII receptor byGDF8. Optionally, an anti-ActRII receptor antibody, or combination ofantibodies, of the disclosure does not substantially inhibit activin Afrom binding to and/or activating an ActRII receptor. In someembodiments, an anti-ActRII receptor antibody, or combination ofantibodies, of the disclosure that binds to an ActRII receptor andprevents binding and/or activation of the ActRII receptor by GDF11and/or GDF8 further prevents binding and/or activation of the ActRIIreceptor by one or more of activin A, activin B, activin AB, activin C,activin E, BMP6, BMP7, and Nodal.

The term antibody is used herein in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity. An antibody fragment refers to amolecule other than an intact antibody that comprises a portion of anintact antibody that binds the antigen to which the intact antibodybinds. Examples of antibody fragments include but are not limited to Fv,Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chainantibody molecules (e.g., scFv); and multispecific antibodies formedfrom antibody fragments. See, e.g., Hudson et al. (2003) Nat. Med.9:129-134; Plückthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); WO 93/16185; and U.S. Pat. Nos. 5,571,894, 5,587,458, and5,869,046. Antibodies disclosed herein may be polyclonal antibodies ormonoclonal antibodies. In certain embodiments, the antibodies of thepresent disclosure comprise a label attached thereto and able to bedetected (e.g., the label can be a radioisotope, fluorescent compound,enzyme, or enzyme co-factor). In preferred embodiments, the antibodiesof the present disclosure are isolated antibodies.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudsonet al. (2003) Nat. Med. 9:129-134 (2003); and Hollinger et al. (1993)Proc. Natl. Acad. Sci. USA 90: 6444-6448. Triabodies and tetrabodies arealso described in Hudson et al. (2003) Nat. Med. 9:129-134.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy-chain variable domain or all or a portion of thelight-chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody. See, e.g.,U.S. Pat. No. 6,248,516.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

The antibodies herein may be of any class. The class of an antibodyrefers to the type of constant domain or constant region possessed byits heavy chain. There are five major classes of antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, andIgA₂. The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu.

In general, an antibody for use in the methods disclosed hereinspecifically binds to its target antigen, preferably with high bindingaffinity. Affinity may be expressed as a K_(D) value and reflects theintrinsic binding affinity (e.g., with minimized avidity effects).Typically, binding affinity is measured in vitro, whether in a cell-freeor cell-associated setting. Any of a number of assays known in the art,including those disclosed herein, can be used to obtain binding affinitymeasurements including, for example, surface plasmon resonance (Biacore™assay), radiolabeled antigen binding assay (MA), and ELISA. In someembodiments, antibodies of the present disclosure bind to their targetantigens (e.g. GDF11, GDF8, ActRIIA, ActRIIB, etc.) with at least aK_(D) of 1×10⁻⁷ or stronger, 1×10⁻⁸ or stronger, 1×10⁻⁹ or stronger,1×10⁻¹⁰ or stronger, 1×10⁻¹¹ or stronger, 1×10⁻¹² or stronger, 1×10⁻¹³or stronger, or 1×10⁻¹⁴ or stronger.

In certain embodiments, K_(D) is measured by MA performed with the Fabversion of an antibody of interest and its target antigen as describedby the following assay. Solution binding affinity of Fabs for theantigen is measured by equilibrating Fab with a minimal concentration ofradiolabeled antigen (e.g., ¹²⁵I-labeled) in the presence of a titrationseries of unlabeled antigen, then capturing bound antigen with ananti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol.Biol. 293:865-881]. To establish conditions for the assay, multi-wellplates (e.g., MICROTITER® from Thermo Scientific) are coated (e.g.,overnight) with a capturing anti-Fab antibody (e.g., from Cappel Labs)and subsequently blocked with bovine serum albumin, preferably at roomtemperature (approximately 23° C.). In a non-adsorbent plate,radiolabeled antigen are mixed with serial dilutions of a Fab ofinterest [e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599]. The Fab ofinterest is then incubated, preferably overnight but the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation, preferably at room temperature for aboutone hour. The solution is then removed and the plate is washed timesseveral times, preferably with polysorbate 20 and PBS mixture. When theplates have dried, scintillant (e.g., MICROSCINT® from Packard) isadded, and the plates are counted on a gamma counter (e.g., TOPCOUNT®from Packard).

According to another embodiment, K_(D) is measured using surface plasmonresonance assays using, for example a BIACORE® 2000 or a BIACORE® 3000(Biacore, Inc., Piscataway, N.J.) with immobilized antigen CM5 chips atabout 10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, Biacore, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions. Forexample, an antigen can be diluted with 10 mM sodium acetate, pH 4.8, to5 μg/ml (about 0.2 μM) before injection at a flow rate of 5 μl/minute toachieve approximately 10 response units (RU) of coupled protein.Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20®) surfactant (PBST) at at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using, for example, a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on) [see, e.g., Chen et al., (1999) J. Mol. Biol.293:865-881]. If the on-rate exceeds, for example, 10⁶ M⁻¹ s⁻¹ by thesurface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (e.g.,excitation=295 nm; emission=340 nm, 16 nm band-pass) of a 20 nManti-antigen antibody (Fab form) in PBS in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO® spectrophotometer (ThermoSpectronic) with a stirred cuvette.

As used herein, anti-GDF11 antibody generally refers to an antibody thatis capable of binding to GDF11 with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingGDF11. In certain embodiments, the extent of binding of an anti-GDF11antibody to an unrelated, non-GDF11 protein is less than about 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of theantibody to GDF11 as measured, for example, by a radioimmunoassay (MA).In certain embodiments, an anti-GDF11 antibody binds to an epitope ofGDF11 that is conserved among GDF11 from different species. In certainpreferred embodiments, an anti-GDF11 antibody of the present disclosureis an antagonist antibody that can inhibit GDF11 activity. For example,an anti-GDF11 antibody of the disclosure may inhibit GDF11 from bindingto a cognate receptor (e.g., ActRIIA or ActRIIB receptor) and/or inhibitGDF11-mediated signal transduction (activation) of a cognate receptor,such as SMAD2/3 signaling by ActRIIA and/or ActRIIB receptors. In someembodiments, anti-GDF11 antibodies of the present disclosure do notsubstantially bind to and/or inhibit activity of activin A. It should benoted that GDF11 has high sequence homology to GDF8 and thereforeantibodies that bind and/or to GDF11, in some cases, may also bind toand/or inhibit GDF8.

An anti-GDF8 antibody refers to an antibody that is capable of bindingto GDF8 with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting GDF8. In certainembodiments, the extent of binding of an anti-GDF8 antibody to anunrelated, non-GDF8 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than 1% of the binding of the antibody to GDF8 asmeasured, for example, by a radioimmunoassay (MA). In certainembodiments, an anti-GDF8 antibody binds to an epitope of GDF8 that isconserved among GDF8 from different species. In preferred embodiments,an anti-GDF8 antibody of the present disclosure is an antagonistantibody that can inhibit GDF8 activity. For example, an anti-GDF8antibody of the disclosure may inhibit GDF8 from binding to a cognatereceptor (e.g., ActRIIA or ActRIIB receptor) and/or inhibitGDF8-mediated signal transduction (activation) of a cognate receptor,such as SMAD2/3 signaling by ActRIIA and/or ActRIIB receptors. In someembodiments, anti-GDF8 antibodies of the present disclosure do notsubstantially bind to and/or inhibit activity of activin A. It should benoted that GDF8 has high sequence homology to GDF11 and thereforeantibodies that bind and/or to GDF8, in many cases, may also bind toand/or inhibit GDF11.

An anti-ActRIIA antibody refers to an antibody that is capable ofbinding to ActRIIA with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting ActRIIA. Incertain embodiments, the extent of binding of an anti-ActRIIA antibodyto an unrelated, non-ActRIIA protein is less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody toActRIIA as measured, for example, by a radioimmunoassay (MA). In certainembodiments, an anti-ActRIIA antibody binds to an epitope of ActRIIAthat is conserved among ActRIIA from different species. In preferredembodiments, an anti-ActRIIA antibody of the present disclosure is anantagonist antibody that can inhibit ActRIIA activity. For example, ananti-ActRIIA antibody of the present disclosure may inhibit one or moreActRIIA ligands selected from activin A, activin B, activin AB, activinC, activin E, GDF11, GDF8, activin A, BMP6, and BMP7 from binding to theActRIIA receptor and/or inhibit one of these ligands from activatingActRIIA signaling (e.g., SMAD2/3 and/or SMAD 1/5/8 ActRIIA signaling).In preferred embodiments, anti-ActRIIA antibodies of the presentdisclosure inhibit GDF11 from binding to the ActRIIA receptor and/orinhibit GDF11 from activating ActRIIA signaling. Optionally,anti-ActRIIA antibodies of the disclosure further inhibit GDF8 frombinding to the ActRIIA receptor and/or inhibit GDF8 from activatingActRIIA signaling. Optionally, anti-ActRIIA antibodies of the presentdisclosure do not substantially inhibit activin A from binding to theActRIIA receptor and/or do not substantially inhibit activin A-mediatedactivation of ActRIIA signaling. In some embodiments, an anti-ActRIIAantibody of the disclosure that inhibits GDF11 and/or GDF8 from bindingto and/or activating an ActRIIA receptor further inhibits one or more ofactivin A, activin B, activin AB, activin C, activin E, activin A, GDF8,BMP6, and BMP7 from binding to and/or activating the ActRIIA receptor.

An anti-ActRIIB antibody refers to an antibody that is capable ofbinding to ActRIIB with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting ActRIIB Incertain embodiments, the extent of binding of an anti-ActRIIB antibodyto an unrelated, non-ActRIIB protein is less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody toActRIIB as measured, for example, by a radioimmunoassay (MA). In certainembodiments, an anti-ActRIIB antibody binds to an epitope of ActRIIBthat is conserved among ActRIIB from different species. In preferredembodiments, an anti-ActRIIB antibody of the present disclosure is anantagonist antibody that can inhibit ActRIIB activity. For example, ananti-ActRIIB antibody of the present disclosure may inhibit one or moreActRIIB ligands selected from activin A, activin B, activin AB, activinC, activin E, GDF11, GDF8, activin A, BMP6, and BMP7 from binding to theActRIIB receptor and/or inhibit one of these ligands from activatingActRIIB signaling (e.g., SMAD2/3 and/or SMAD 1/5/8 ActRIIB signaling).In preferred embodiments, anti-ActRIIB antibodies of the presentdisclosure inhibit GDF11 from binding to the ActRIIB receptor and/orinhibit GDF11 from activating ActRIIB signaling. Optionally,anti-ActRIIB antibodies of the disclosure further inhibit GDF8 frombinding to the ActRIIB receptor and/or inhibit GDF8 from activatingActRIIB signaling. Optionally, anti-ActRIIB antibodies of the presentdisclosure do not substantially inhibit activin A from binding to theActRIIB receptor and/or do not substantially inhibit activin A-mediatedactivation of ActRIIB signaling. In some embodiments, an anti-ActRIIBantibody of the disclosure that inhibits GDF11 and/or GDF8 from bindingto and/or activating an ActRIIB receptor further inhibits one or more ofactivin A, activin B, activin AB, activin C, activin E, activin A, GDF8,BMP6, and BMP7 from binding to and/or activating the ActRIIB receptor.

The nucleic acid and amino acid sequences of human GDF11, GDF8, activinA, activin B, activin AB, activin C, activin E, GDF8, BMP6, BMP7,ActRIIB, and ActRIIA are well known in the art and thus antibodyantagonists for use in accordance with this disclosure may be routinelymade by the skilled artisan based on the knowledge in the art andteachings provided herein.

In certain embodiments, an antibody provided herein (e.g., an anti-GDF11antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, or ananti-ActRIIB antibody) is a chimeric antibody. A chimeric antibodyrefers to an antibody in which a portion of the heavy and/or light chainis derived from a particular source or species, while the remainder ofthe heavy and/or light chain is derived from a different source orspecies. Certain chimeric antibodies are described, for example, in U.S.Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci.USA, 81:6851-6855. In some embodiments, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In some embodiments, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. In general, chimericantibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody provided herein (e.g., ananti-GDF11 antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, oran anti-ActRIIB antibody) is a humanized antibody. A humanized antibodyrefers to a chimeric antibody comprising amino acid residues fromnon-human hypervariable regions (HVRs) and amino acid residues fromhuman framework regions (FRs). In certain embodiments, a humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the HVRs(e.g., CDRs) correspond to those of a non-human antibody, and all orsubstantially all of the FRs correspond to those of a human antibody. Ahumanized antibody optionally may comprise at least a portion of anantibody constant region derived from a human antibody. A “humanizedform” of an antibody, e.g., a non-human antibody, refers to an antibodythat has undergone humanization.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633 andare further described, for example, in Riechmann et al., (1988) Nature332:323-329; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri et al., (2005) Methods 36:25-34 [describing SDR(a-CDR) grafting]; Padlan, Mol. Immunol. (1991) 28:489-498 (describing“resurfacing”); Dall'Acqua et al. (2005) Methods 36:43-60 (describing“FR shuffling”); Osbourn et al. (2005) Methods 36:61-68; and Klimka etal. Br. J. Cancer (2000) 83:252-260 (describing the “guided selection”approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method [see, e.g., Sims et al. (1993) J. Immunol. 151:2296]; frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light-chain or heavy-chain variable regions [see,e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; andPresta et al. (1993) J. Immunol., 151:2623]; human mature (somaticallymutated) framework regions or human germline framework regions [see,e.g., Almagro and Fransson (2008) Front. Biosci. 13:1619-1633]; andframework regions derived from screening FR libraries [see, e.g., Bacaet cd., (1997) J. Biol. Chem. 272:10678-10684; and Rosok et cd., (1996)J. Biol. Chem. 271:22611-22618].

In certain embodiments, an antibody provided herein (e.g., an anti-GDF11antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, or ananti-ActRIIB antibody) is a human antibody. Human antibodies can beproduced using various techniques known in the art. Human antibodies aredescribed generally in van Dijk and van de Winkel (2001) Curr. Opin.Pharmacol. 5: 368-74 and Lonberg (2008) Curr. Opin. Immunol. 20:450-459.

Human antibodies may be prepared by administering an immunogen (e.g., aGDF11 polypeptide, GDF8 polypeptide, an ActRIIA polypeptide, or anActRIIB polypeptide) to a transgenic animal that has been modified toproduce intact human antibodies or intact antibodies with human variableregions in response to antigenic challenge. Such animals typicallycontain all or a portion of the human immunoglobulin loci, which replacethe endogenous immunoglobulin loci, or which are presentextrachromosomally or integrated randomly into the animal's chromosomes.In such transgenic animals, the endogenous immunoglobulin loci havegenerally been inactivated. For a review of methods for obtaining humanantibodies from transgenic animals, see, for example, Lonberg (2005)Nat. Biotechnol. 23:1117-1125; U.S. Pat. Nos. 6,075,181 and 6,150,584(describing XENOMOUSE™ technology); U.S. Pat. No. 5,770,429 (describingHuMab® technology); U.S. Pat. No. 7,041,870 (describing K-M MOUSE®technology); and U.S. Patent Application Publication No. 2007/0061900(describing VelociMouse® technology). Human variable regions from intactantibodies generated by such animals may be further modified, forexample, by combining with a different human constant region.

Human antibodies provided herein can also be made by hybridoma-basedmethods. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described [see,e.g., Kozbor J. Immunol., (1984) 133: 3001; Brodeur et al. (1987)Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York; and Boerner et al. (1991) J. Immunol.,147: 86]. Human antibodies generated via human B-cell hybridomatechnology are also described in Li et al., (2006) Proc. Natl. Acad.Sci. USA, 103:3557-3562. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 (describing production of monoclonalhuman IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue (2006) 26(4):265-268 (2006) (describing human-humanhybridomas). Human hybridoma technology (Trioma technology) is alsodescribed in Vollmers and Brandlein (2005) Histol. Histopathol.,20(3):927-937 (2005) and Vollmers and Brandlein (2005) Methods Find Exp.Clin. Pharmacol., 27(3):185-91.

Human antibodies provided herein (e.g., an anti-GDF11 antibody, ananti-activin B antibody, an anti-ActRIIA antibody, or an anti-ActRIIBantibody) may also be generated by isolating Fv clone variable-domainsequences selected from human-derived phage display libraries. Suchvariable-domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described herein.

For example, antibodies of the present disclosure may be isolated byscreening combinatorial libraries for antibodies with the desiredactivity or activities. A variety of methods are known in the art forgenerating phage-display libraries and screening such libraries forantibodies possessing the desired binding characteristics. Such methodsare reviewed, for example, in Hoogenboom et al. (2001) in Methods inMolecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa,N.J. and further described, for example, in the McCafferty et al. (1991)Nature 348:552-554; Clackson et al., (1991) Nature 352: 624-628; Markset al. (1992) J. Mol. Biol. 222:581-597; Marks and Bradbury (2003) inMethods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa,N.J.; Sidhu et al. (2004) J. Mol. Biol. 338(2):299-310; Lee et al.(2004) J. Mol. Biol. 340(5):1073-1093; Fellouse (2004) Proc. Natl. Acad.Sci. USA 101(34):12467-12472; and Lee et al. (2004) J. Immunol. Methods284(1-2): 119-132.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al. (1994) Ann. Rev.Immunol., 12: 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen (e.g., GDF11, activin B, ActRIIA, or ActRIIB) without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned (e.g., from human) to provide a single sourceof antibodies directed against a wide range of non-self and alsoself-antigens without any immunization as described by Griffiths et al.(1993) EMBO J, 12: 725-734. Finally, naive libraries can also be madesynthetically by cloning un-rearranged V-gene segments from stem cellsand using PCR primers containing random sequence to encode the highlyvariable CDR3 regions and to accomplish rearrangement in vitro, asdescribed by Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388.Patent publications describing human antibody phage libraries include,for example: U.S. Pat. No. 5,750,373, and U.S. Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodies(typically monoclonal antibodies) have binding specificities for atleast two different epitopes (e.g., two, three, four, five, or six ormore) on one or more (e.g., two, three, four, five, six or more)antigens.

In certain embodiments, a multispecific antibody of the presentdisclosure comprises two or more binding specificities, with at leastone of the binding specificities being for a GDF11 epitope, andoptionally one or more additional binding specificities being for anepitope on a different ActRII ligand (e.g., GDF8, activin A, activin B,activin AB, activin C, activin E, BMP6 BMP7 and/or Nodal) and/or anActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor). In certainembodiments, multispecific antibodies may bind to two or more differentepitopes of GDF11. Preferably a multispecific antibody of the disclosurethat has binding affinity, in part, for a GDF11 epitope can be used toinhibit a GDF11 activity (e.g., the ability to bind to and/or activatean ActRIIA and/or ActRIIB receptor), and optionally inhibit the activityof one or more different ActRII ligands (e.g., GDF8, activin A, activinB, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or anActRII receptor (e.g., an ActRIIA or ActRIIB receptor). In certainpreferred embodiments, multispecific antibodies of the presentdisclosure that bind to and/or inhibit GDF11 further bind to and/orinhibit at least GDF8. Optionally, multispecific antibodies of thedisclosure that bind to and/or inhibit GDF11 do not substantially bindto and/or substantially inhibit activin A. In some embodiments,multispecific antibodies of the disclosure that bind to and/or inhibitGDF11 and GDD8 further bind to and/or inhibit one or more of activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal.

In certain embodiments, a multispecific antibody of the presentdisclosure comprises two or more binding specificities, with at leastone of the binding specificities being for a GDF8 epitope, andoptionally one or more additional binding specificities being for anepitope on a different ActRII ligand (e.g., GDF11, activin A, activin B,activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or anActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor). In certainembodiments, multispecific antibodies may bind to two or more differentepitopes of GDF8. Preferably a multispecific antibody of the disclosurethat has binding affinity, in part, for an GDF8 epitope can be used toinhibit an GDF8 activity (e.g., the ability to bind to and/or activatean ActRIIA and/or ActRIIB receptor), and optionally inhibit the activityof one or more different ActRII ligands (e.g., GDF11, activin A, activinB, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or anActRII receptor (e.g., an ActRIIA or ActRIIB receptor). In certainpreferred embodiments, multispecific antibodies of the presentdisclosure that bind to and/or inhibit GDF8 further bind to and/orinhibit at least GDF11. Optionally, multispecific antibodies of thedisclosure that bind to and/or inhibit GDF8 do not substantially bind toand/or substantially inhibit activin A. In some embodiments,multispecific antibodies of the disclosure that bind to and/or inhibitGDF8 and GDF11 further bind to and/or inhibit one or more of activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal.

Engineered antibodies with three or more functional antigen bindingsites, including “octopus antibodies,” are also included herein (see,e.g., US 2006/0025576A1).

In certain embodiments, the antibodies disclosed herein (e.g., ananti-GDF11 antibody, an anti-activin B antibody, an anti-ActRIIAantibody, or an anti-ActRIIB antibody) are monoclonal antibodies.Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical and/or bind the same epitope,except for possible variant antibodies, e.g., containing naturallyoccurring mutations or arising during production of a monoclonalantibody preparation, such variants generally being present in minoramounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentepitopes, each monoclonal antibody of a monoclonal antibody preparationis directed against a single epitope on an antigen. Thus, the modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present methods may be made by a variety of techniques,including but not limited to the hybridoma method, recombinant DNAmethods, phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein.

For example, by using immunogens derived from GDF11 or GDF8,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols [see, e.g., Antibodies: A Laboratory Manual (1988)ed. by Harlow and Lane, Cold Spring Harbor Press]. A mammal, such as amouse, hamster, or rabbit can be immunized with an immunogenic form ofthe GDF11 or GDF8 polypeptide, an antigenic fragment which is capable ofeliciting an antibody response, or a fusion protein. Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers or other techniques well known in the art. An immunogenicportion of a GDF11 or GDF8 polypeptide can be administered in thepresence of adjuvant. The progress of immunization can be monitored bydetection of antibody titers in plasma or serum. Standard ELISA or otherimmunoassays can be used with the immunogen as antigen to assess thelevels of antibody production and/or level of binding affinity.

Following immunization of an animal with an antigenic preparation ofGDF11 or GDF8, antisera can be obtained and, if desired, polyclonalantibodies can be isolated from the serum. To produce monoclonalantibodies, antibody-producing cells (lymphocytes) can be harvested froman immunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique [see, e.g., Kohler and Milstein (1975) Nature, 256:495-497], the human B cell hybridoma technique [see, e.g., Kozbar et al.(1983) Immunology Today, 4:72], and the EBV-hybridoma technique toproduce human monoclonal antibodies [Cole et al. (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96]. Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with a GDF11 or GDF8 polypeptide, and monoclonalantibodies isolated from a culture comprising such hybridoma cells.

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein (e.g., ananti-GDF11 antibody, an anti-activin B antibody, an anti-ActRIIAantibody, or an anti-ActRIIB antibody), thereby generating an Fc-regionvariant. The Fc-region variant may comprise a human Fc-region sequence(e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an aminoacid modification (e.g., a substitution, deletion, and/or addition) atone or more amino acid positions.

For example, the present disclosure contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet for which certain effector functions[e.g., complement-dependent cytotoxicity (CDC) and antibody-dependentcellular cytotoxicity (ADCC)] are unnecessary or deleterious. In vitroand/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in, forexample, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362;Hellstrom, I. et al. (1986) Proc. Nat'l Acad. Sci. USA 83:7059-7063;Hellstrom, I et al. (1985) Proc. Nat'l Acad. Sci. USA 82:1499-1502; U.S.Pat. No. 5,821,337; and Bruggemann, M. et al. (1987) J. Exp. Med.166:1351-1361. Alternatively, non-radioactive assay methods may beemployed (e.g., ACTI™, non-radioactive cytotoxicity assay for flowcytometry; CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay, Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, for example, in an animal model such as that disclosed inClynes et al. (1998) Proc. Nat'l Acad. Sci. USA 95:652-656. C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity [see, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402]. To assess complementactivation, a CDC assay may be performed [see, e.g., Gazzano-Santoro etal. (1996) J. Immunol. Methods 202:163; Cragg, M. S. et al. (2003) Blood101:1045-1052; and Cragg, M. S, and M. J. Glennie (2004) Blood103:2738-2743]. FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art[see, e.g., Petkova, S. B. et al. (2006) Int. Immunol.18(12):1759-1769].

Antibodies of the present disclosure (e.g., an anti-GDF11 antibody, ananti-activin B antibody, an anti-ActRITA antibody, or an anti-ActRIIBantibody) with reduced effector function include those with substitutionof one or more of Fc region residues 238, 265, 269, 270, 297, 327 and329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

In certain embodiments, it may be desirable to createcysteine-engineered antibodies, e.g., “thioMAbs,” in which one or moreresidues of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to other moieties,such as drug moieties or linker-drug moieties, to create animmunoconjugate, as described further herein. In certain embodiments,any one or more of the following residues may be substituted withcysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering)of the heavy chain; and 5400 (EU numbering) of the heavy-chain Fcregion. Cysteine engineered antibodies may be generated as described,for example, in U.S. Pat. No. 7,521,541.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore™ binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain embodiments, amino acid sequence variants of the antibodiesand/or the binding polypeptides provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the antibody and/or binding polypeptide.Amino acid sequence variants of an antibody and/or binding polypeptidesmay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody and/or binding polypeptide, orby peptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into, and/or substitutions of residues within,the amino acid sequences of the antibody and/or binding polypeptide. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., target-binding (GDF11,GDF8, ActRIIA, and/or ActRIIB binding).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process (see, e.g.,Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs(a-CDRs), with the resulting variant VH or VL being tested for bindingaffinity. Affinity maturation by constructing and reselecting fromsecondary libraries has been described in the art [see, e.g., Hoogenboomet al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed.,Human Press, Totowa, N.J., (2001)]. In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind to the antigen.For example, conservative alterations (e.g., conservative substitutionsas provided herein) that do not substantially reduce binding affinitymay be made in HVRs. Such alterations may be outside of HVR “hotspots”or SDRs. In certain embodiments of the variant VH and VL sequencesprovided above, each HVR either is unaltered, or contains no more thanone, two, or three amino acid substitutions.

A useful method for identification of residues or regions of theantibody and/or the binding polypeptide that may be targeted formutagenesis is called “alanine scanning mutagenesis”, as described byCunningham and Wells (1989) Science, 244:1081-1085. In this method, aresidue or group of target residues (e.g., charged residues such as arg,asp, his, lys, and glu) are identified and replaced by a neutral ornegatively charged amino acid (e.g., alanine or polyalanine) todetermine whether the interaction of the antibody or binding polypeptidewith antigen is affected. Further substitutions may be introduced at theamino acid locations demonstrating functional sensitivity to the initialsubstitutions. Alternatively, or additionally, a crystal structure of anantigen-antibody complex can be used to identify contact points betweenthe antibody and antigen. Such contact residues and neighboring residuesmay be targeted or eliminated as candidates for substitution. Variantsmay be screened to determine whether they contain the desiredproperties.

Amino-acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include fusion of the N- or C-terminusof the antibody to an enzyme (e.g., for ADEPT) or a polypeptide whichincreases the serum half-life of the antibody.

In certain embodiments, an antibody and/or binding polypeptide providedherein may be further modified to contain additional non-proteinaceousmoieties that are known in the art and readily available. The moietiessuitable for derivatization of the antibody and/or binding polypeptideinclude but are not limited to water-soluble polymers. Non-limitingexamples of water-soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody and/orbinding polypeptide may vary, and if more than one polymer are attached,they can be the same or different molecules. In general, the numberand/or type of polymers used for derivatization can be determined basedon considerations including, but not limited to, the particularproperties or functions of the antibody and/or binding polypeptide to beimproved, whether the antibody derivative and/or binding polypeptidederivative will be used in a therapy under defined conditions.

Any of the ActRII antagonist antibodies disclosed herein (e.g., ananti-activin A antibody, an anti-activin B antibody, an anti-activin Cantibody, an anti-activin E antibody, an anti-GDF11 antibody, ananti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, ananti-ActRIIA antibody, and/or or an anti-ActRIIB antibody) can becombined with one or more additional ActRII antagonist agents of thedisclosure to achieve the desired effect. For example, an ActRIIantagonist antibody disclosed herein (e.g., an anti-GDF11 antibody, ananti-activin B antibody, an anti-activin C antibody, an anti-activin Eantibody, an anti-GDF11 antibody, an anti-GDF8 antibody, an anti-BMP6antibody, an-anti-BMP7 antibody, an anti-ActRIIA antibody, or ananti-ActRIIB antibody) can be used in combination with i) one or moreadditional ActRII antagonist antibodies disclosed herein, ii) one ormore ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIBpolypeptides), iii) one or more GDF traps disclosed herein; iv) one ormore small-molecule ActRII antagonist disclosed herein (e.g., a smallmolecule antagonist of one or more of GDF11, GDF8, activin A, activin B,activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/orActRIIB); v) one or more polynucleotide ActRII antagonists disclosedherein (e.g., a polynucleotide antagonist of one or more of GDF11, GDF8,activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7,Nodal, ActRIIA, and/or ActRIIB); vi) one or more follistatinpolypeptides disclosed herein; and/or vii) one or more FLRG polypeptidesdisclosed herein.

D. Small-Molecule Antagonists

In another aspect, the present disclosure relates to a small molecule,or combination of small molecules, that antagonizes ActRII activity(e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, suchas SMAD 2/3 and/or SMAD 1/5/8 signaling). In particular, the disclosureprovides methods of using a small-molecule antagonist, or combination ofantibody antagonists, of ActRII, alone or in combination with one ormore erythropoiesis stimulating agents (e.g., EPO) or other supportivetherapies [e.g., hematopoietic growth factors (e.g., G-CSF or GM-CSF),transfusion of red blood cells or whole blood, iron chelation therapy],to, e.g., increase red blood cell levels in a subject in need thereof,treat or prevent an anemia in a subject in need thereof (including,e.g., reduction of transfusion burden), treat MDS or sideroblasticanemias in a subject in need thereof, and/or treat or prevent one ormore complications of MDS or sideroblastic anemias (e.g., anemia, bloodtransfusion requirement, neutropenia, iron overload, acute myocardialinfarction, hepatic failure, hepatomegaly, splenomegaly, progression toacute myeloid lymphoma) and or treat or prevent a disorder associatedwith SF3B1, DNMT3A, and/or TET2 mutations in a subject in need thereof.

In some embodiments, a preferred ActRII antagonist of the presentdisclosure is a small-molecule antagonist, or combination ofsmall-molecule antagonists, that direct or indirect inhibits at leastGDF11 activity. Optionally, such a small-molecule antagonist, orcombination of small-molecule antagonists, may further inhibit, eitherdirectly or indirectly, GDF8. Optionally, a small-molecule antagonist,or combination of small-molecule antagonists, of the present disclosuredoes not substantially inhibit activin A activity. In some embodiments,a small-molecule antagonist, or combination of small-moleculeantagonists, of the present disclosure that inhibits, either directly orindirectly, GDF11 and/or GDF8 activity further inhibits, either directlyor indirectly, activity of one or more of activin A, activin B, activinAB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB

In certain embodiments, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the present disclosure is an indirectinhibitor of one or more of GDF11, GDF8, activin A, activin B, activinAB, activin C, activin E, BMP6, Nodal, ActRIIA, and ActRIIB For example,a small-molecule antagonist, or combination of small-moleculeantagonists, of the present disclosure may inhibit the expression (e.g.,transcription, translation, cellular secretion, or combinations thereof)of at least GDF11. Optionally, such a small-molecule antagonist, orcombination of small-molecule antagonists, may further inhibitexpression of GDF8. Optionally, a small-molecule antagonist, orcombinations of small-molecule antagonists, of the disclosure does notsubstantially inhibit the expression of activin A. In some embodiments,a small-molecule antagonist, or combination of small-moleculeantagonists, of the disclosure that inhibits expression of GDF11 and/orGDF8 may further inhibit the expression of one or more of activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and ActRIIB

In other embodiments, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the present disclosure is directinhibitor of one or more of GDF11, GDF8, activin A, activin B, activinAB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB Forexample, a preferred small-molecule antagonist, or combination ofsmall-molecule antagonists, of the present disclosure directly binds toand inhibits at least GDF11 activity (e.g. inhibits the ability GDF11 tobind to an ActRIIA and/or ActRIIB receptor; inhibits GDF11-mediatedactivation of the ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 signaling). Optionally, a small-molecule antagonist, orcombinations of small-molecule antagonists, of the disclosure mayfurther bind to and inhibit GDF8 activity (e.g. inhibits the ability ofGDF8 to bind to an ActRIIA and/or ActRIIB receptor; inhibitsGDF8-mediated activation of the ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling). Optionally, a small-moleculeantagonist, or combinations of small-molecule antagonists, of thedisclosure does not substantially bind to or inhibit activin A activity(e.g. the ability of activin A to bind to an ActRIIA and/or ActRIIBreceptor; activin A-mediated activation of the ActRIIA and/or ActRIIBsignaling transduction, such as SMAD 2/3 signaling pathway). In someembodiments, a small-molecule antagonist, or combinations ofsmall-molecule antagonists, of the disclosure that binds to and inhibitsthe activity of GDF11 and/or GDF8 further binds to and inhibits theactivity of one or more of activin A, activin B, activin AB, activin C,activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB

In some embodiments, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the present disclosure directly binds toand inhibits at least GDF8 activity (e.g. inhibits the ability GDF8 tobind to an ActRIIA and/or ActRIIB receptor; inhibits GDF8-mediatedactivation of the ActRIIA and/or ActRIIB signaling transduction, such asSMAD 2/3 signaling). Optionally, a small-molecule antagonist, orcombinations of small-molecule antagonists, of the disclosure mayfurther bind to and inhibit GDF11 activity (e.g. inhibit the ability ofGDF11 to bind to an ActRIIA and/or ActRIIB receptor; inhibitGDF11-mediated activation of the ActRIIA and/or ActRIIB signalingtransduction, such as SMAD 2/3 signaling). Optionally, a small-moleculeantagonist, or combinations of small-molecule antagonists, of thedisclosure does not substantially bind to or inhibit activin A activity(e.g. the ability of activin A to bind to an ActRIIA and/or ActRIIBreceptor; activin A-mediated activation of the ActRIIA and/or ActRIIBsignaling transduction, SMAD 2/3 signaling). In some embodiments, asmall-molecule antagonist, or combinations of small-moleculeantagonists, of the disclosure that binds to and inhibits the activityof GDF8 and/or GDF11 further binds to and inhibits the activity of oneor more of activin A, activin B, activin AB, activin C, activin E, BMP6,BMP7, Nodal, ActRIIA, and ActRIIB

In some embodiments, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the present disclosure directly binds toand inhibits at least ActRIIA activity (e.g. ActRII ligand-mediatedactivation of ActRIIA signaling transduction, such as SMAD 2/3signaling). For example, a preferred small-molecule antagonist, orcombination of small-molecule antagonists, of the disclosure binds to anActRIIA receptor and inhibits at least GDF11 from binding to and/oractivating the ActRIIA receptor. Optionally, such a small-moleculeantagonist, or combination of small-molecule antagonists, may furtherinhibit GDF8 from binding to and/or activating the ActRIIA receptor.Optionally, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the disclosure does not substantiallyinhibit activin A from binding to and/or activating an ActRIIA receptor.In some embodiments, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the disclosure that inhibits GDF11 and/orGDF8 from binding to and/or activating the ActRIIA receptor furtherinhibits one or more of activin A, activin B, activin AB, activin C,activin E, BMP6, BMP7, and Nodal from binding to/and or activating theActRIIA receptor.

In some embodiments, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the present disclosure directly binds toand inhibits at least ActRIIB activity (e.g. ActRII ligand-mediatedactivation of ActRIIB signaling transduction, such as SMAD 2/3signaling). For example, a preferred small-molecule antagonist, orcombination of small-molecule antagonists, of the disclosure binds to anActRIIB receptor and inhibits at least GDF11 from binding to and/oractivating the ActRIIB receptor. Optionally, such a small-moleculeantagonist, or combination of small-molecule antagonists, may furtherinhibit GDF8 from binding to and/or activating the ActRIIB receptor.Optionally, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the disclosure does not substantiallyinhibit activin A from binding to and/or activating an ActRIIB receptor.In some embodiments, a small-molecule antagonist, or combination ofsmall-molecule antagonists, of the disclosure that inhibits GDF11 and/orGDF8 from binding to and/or activating the ActRIIB receptor furtherinhibits one or more of activin A, activin B, activin AB, activin C,activin E, BMP6, BMP7, and Nodal from binding to/and or activating theActRIIB receptor.

Binding organic small molecule antagonists of the present disclosure maybe identified and chemically synthesized using known methodology (see,e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585). In general,small-molecule antagonists of the disclosure are usually less than about2000 daltons in size, alternatively less than about 1500, 750, 500, 250or 200 daltons in size, wherein such organic small molecules that arecapable of binding, preferably specifically, to a polypeptide asdescribed herein (e.g., GDF11, GDF8, ActRIIA, and ActRIIB) Suchsmall-molecule antagonists may be identified without undueexperimentation using well-known techniques. In this regard, it is notedthat techniques for screening organic small-molecule libraries formolecules that are capable of binding to a polypeptide target arewell-known in the art (see, e.g., international patent publication Nos.WO00/00823 and WO00/39585).

Binding organic small molecules of the present disclosure may be, forexample, aldehydes, ketones, oximes, hydrazones, semicarbazones,carbazides, primary amines, secondary amines, tertiary amines,N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,thioethers, disulfides, carboxylic acids, esters, amides, ureas,carbamates, carbonates, ketals, thioketals, acetals, thioacetals, arylhalides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromaticcompounds, heterocyclic compounds, anilines, alkenes, alkynes, diols,amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds, and acid chlorides.

Any of the small-molecule ActRII antagonists disclosed herein (e.g., asmall-molecule antagonist of one or more of GDF11, GDF8, activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and/or ActRIIB) can be combined with one or more additional ActRIIantagonist agents of the disclosure to achieve the desired effect (e.g.,increase red blood cell levels and/or hemoglobin in a subject in needthereof, treat or prevent an anemia, treat MDS or sideroblastic anemias,treat or prevent one or more complications of MDS or sideroblasticanemias). For example, a small-molecule ActRII antagonist disclosedherein (e.g., a small-molecule antagonist of one or more of GDF11, GDF8,activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7,Nodal, ActRIIA, and/or ActRIIB) can be used in combination with i) oneor more additional small molecule ActRII antagonists disclosed herein,ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIAand/or ActRIIB polypeptides), iii) one or more GDF traps disclosedherein; iv) one or more ActRII antagonist antibodies disclosed herein(e.g., an anti-GDF11 antibody, an anti-activin B antibody, ananti-activin C antibody, an anti-activin E antibody, an anti-GDF11antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody); v) oneor more polynucleotide ActRII antagonists disclosed herein (e.g., apolynucleotide antagonist of one or more of GDF11, GDF8, activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and/or ActRIIB); vi) one or more follistatin polypeptides disclosedherein; and/or vii) one or more FLRG polypeptides disclosed herein.

E. Antagonist Polynucleotides

In another aspect, the present disclosure relates to a polynucleotide,or combination of polynucleotides, that antagonizes ActRII activity(e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, suchas SMAD 2/3 and/or SMAD 1/5/8 signaling). In particular, the disclosureprovides methods of using a polynucleotide ActRII antagonist, orcombination of polynucleotide ActRII antagonists, alone or incombination with one or more erythropoiesis stimulating agents (e.g.,EPO) or other supportive therapies [e.g., hematopoietic growth factors(e.g., G-CSF or GM-CSF), transfusion of red blood cells or whole blood,iron chelation therapy], to, e.g., increase red blood cell levels in asubject in need thereof, treat or prevent an anemia in a subject in needthereof (including, e.g., reduction of transfusion burden), treat MDS orsideroblastic anemias in a subject in need thereof, and/or treat orprevent one or more complications of MDS or sideroblastic anemias (e.g.,anemia, blood transfusion requirement, neutropenia, iron overload, acutemyocardial infarction, hepatic failure, hepatomegaly, splenomegaly,progression to acute myeloid lymphoma) and or treat or prevent adisorder associated with SF3B1, DNMT3A, and/or TET2 mutations in asubject in need thereof.

In some embodiments, a polynucleotide ActRII antagonist, or combinationof polynucleotide ActRII antagonists, of the present disclosure can beused to inhibit the activity and/or expression on one or more of GDF11,GDF8, activin A, activin B, activin AB, activin C, activin C, activin E,BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB In certain preferredembodiments, a polynucleotide ActRII antagonist, or combination ofpolynucleotide ActRII antagonists, of the disclosure is a GDF-ActRIIantagonist.

In some embodiments, a polynucleotide antagonist, or combination ofpolynucleotide antagonists, of the disclosure inhibits the activityand/or expression (e.g., transcription, translation, secretion, orcombinations thereof) of at least GDF11. Optionally, such apolynucleotide antagonist, or combination of polynucleotide antagonists,may further inhibit the activity and/or expression of GDF8. Optionally,a polynucleotide antagonist, or combination of polynucleotideantagonists, of the disclosure does not substantially inhibit theactivity and/or expression of activin A. In some embodiments, apolynucleotide antagonist, or combination of polynucleotide antagonists,of the disclosure that inhibits the activity and/or expression of GDF11and/or GDF8 may further inhibit the activity and or expression of one ormore of activin A, activin B, activin AB, activin C, activin E, BMP6,BMP7, Nodal, ActRIIA, and/or ActRIIB

In some embodiments, a polynucleotide antagonist, or combination ofpolynucleotide antagonists, of the disclosure inhibits the activityand/or expression (e.g., transcription, translation, secretion, orcombinations thereof) of at least GDF8. Optionally, such polynucleotideantagonist, or combination of polynucleotide antagonists, may furtherinhibit the activity and/or expression of GDF11. Optionally, apolynucleotide antagonist, or combination of polynucleotide antagonists,of the disclosure does not substantially inhibit the activity and/orexpression of activin A. In some embodiments, a polynucleotideantagonist, or combination of polynucleotide antagonists, of thedisclosure that inhibits the activity and/or expression of GDF8 and/orGDF11 may further inhibit the activity and or expression of one or moreof activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7,Nodal, ActRIIA, and/or ActRIIB

In some embodiments, a polynucleotide antagonist, or combination ofpolynucleotide antagonists, of the disclosure inhibits the activityand/or expression (e.g., transcription, translation, secretion, orcombinations thereof) of at least ActRIIA. Optionally, a polynucleotideantagonist, or combination of polynucleotide antagonists, of thedisclosure does not substantially inhibit the activity and/or expressionof activin A. In some embodiments, a polynucleotide antagonist, orcombination of polynucleotide antagonists, of the disclosure thatinhibits the activity and/or expression of ActRIIA may further inhibitthe activity and or expression of one or more of activin A, activin B,activin AB, activin C, activin E, BMP6, BMP7, Nodal, and/or ActRIIB

In some embodiments, a polynucleotide antagonist, or combination ofpolynucleotide antagonists, of the disclosure inhibits the activityand/or expression (e.g., transcription, translation, secretion, orcombinations thereof) of at least ActRIIB Optionally, a polynucleotideantagonist, or combination of polynucleotide antagonists, of thedisclosure does not substantially inhibit the activity and/or expressionof activin A. In some embodiments, a polynucleotide antagonist, orcombination of polynucleotide antagonists, of the disclosure thatinhibits the activity and/or expression of ActRIIB may further inhibitthe activity and or expression of one or more of activin A, activin B,activin AB, activin C, activin E, BMP6, BMP7, Nodal, and/or ActRIIA.

The polynucleotide antagonists of the present disclosure may be anantisense nucleic acid, an RNAi molecule [e.g., small interfering RNA(siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)], an aptamer and/ora ribozyme. The nucleic acid and amino acid sequences of human GDF11,GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal,ActRIIA, and ActRIIB are known in the art and thus polynucleotideantagonists for use in accordance with methods of the present disclosuremay be routinely made by the skilled artisan based on the knowledge inthe art and teachings provided herein.

For example, antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed, for example, in Okano (1991) J.Neurochem. 56:560; Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Cooney et al. (1988) Science 241:456; andDervan et al., (1991) Science 251:1300. The methods are based on bindingof a polynucleotide to a complementary DNA or RNA. In some embodiments,the antisense nucleic acids comprise a single-stranded RNA or DNAsequence that is complementary to at least a portion of an RNAtranscript of a gene disclosed herein (e.g., GDF11, GDF8, activin A,activin B, activin C, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and ActRIIB) However, absolute complementarity, although preferred, isnot required.

A sequence “complementary to at least a portion of an RNA,” referred toherein, means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex; in the case ofdouble-stranded antisense nucleic acids of a gene disclosed herein(e.g., GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6,BMP7, Nodal, ActRIIA, and ActRIIB), a single strand of the duplex DNAmay thus be tested, or triplex formation may be assayed. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Generally, the larger thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Polynucleotides that are complementary to the 5′ end of the message, forexample, the 5′-untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′-untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well [see, e.g., Wagner, R., (1994) Nature372:333-335]. Thus, oligonucleotides complementary to either the 5′- or3′-untranslated, noncoding regions of a gene of the disclosure (e.g.,GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7,Nodal, ActRIIA, and ActRIIB), could be used in an antisense approach toinhibit translation of an endogenous mRNA. Polynucleotides complementaryto the 5′-untranslated region of the mRNA should include the complementof the AUG start codon. Antisense polynucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the methods of the present disclosure. Whetherdesigned to hybridize to the 5′-untranslated, 3′-untranslated, or codingregions of an mRNA of the disclosure (e.g., an GDF11, GDF8, activin A,activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIBmRNA), antisense nucleic acids should be at least six nucleotides inlength, and are preferably oligonucleotides ranging from 6 to about 50nucleotides in length. In specific aspects, the oligonucleotide is atleast 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides,or at least 50 nucleotides.

In one embodiment, the antisense nucleic acid of the present disclosure(e.g., a GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6,BMP7, Nodal, ActRIIA, or ActRIIB antisense nucleic acid) is producedintracellularly by transcription from an exogenous sequence. Forexample, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of a gene of the disclosure. Such a vectorwould contain a sequence encoding the desired antisense nucleic acid.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding desired genes of the instantdisclosure, or fragments thereof, can be by any promoter known in theart to act in vertebrate, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include, but are not limitedto, the SV40 early promoter region [see, e.g., Benoist and Chambon(1981) Nature 29:304-310], the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al. (1980)Cell 22:787-797], the herpes thymidine promoter [see, e.g., Wagner etal. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445], and theregulatory sequences of the metallothionein gene [see, e.g., Brinster,et al. (1982) Nature 296:39-42].

In some embodiments, the polynucleotide antagonists are interfering RNAor RNAi molecules that target the expression of one or more of: GDF11,GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal,ActRIIA, and ActRIIB RNAi refers to the expression of an RNA whichinterferes with the expression of the targeted mRNA. Specifically, RNAisilences a targeted gene via interacting with the specific mRNA througha siRNA (small interfering RNA). The ds RNA complex is then targeted fordegradation by the cell. An siRNA molecule is a double-stranded RNAduplex of 10 to 50 nucleotides in length, which interferes with theexpression of a target gene which is sufficiently complementary (e.g. atleast 80% identity to the gene). In some embodiments, the siRNA moleculecomprises a nucleotide sequence that is at least 85, 90, 95, 96, 97, 98,99, or 100% identical to the nucleotide sequence of the target gene.

Additional RNAi molecules include short-hairpin RNA (shRNA); alsoshort-interfering hairpin and microRNA (miRNA). The shRNA moleculecontains sense and antisense sequences from a target gene connected by aloop. The shRNA is transported from the nucleus into the cytoplasm, andit is degraded along with the mRNA. Pol III or U6 promoters can be usedto express RNAs for RNAi. Paddison et al. [Genes & Dev. (2002)16:948-958, 2002] have used small RNA molecules folded into hairpins asa means to effect RNAi. Accordingly, such short hairpin RNA (shRNA)molecules are also advantageously used in the methods described herein.The length of the stem and loop of functional shRNAs varies; stemlengths can range anywhere from about 25 to about 30 nt, and loop sizecan range between 4 to about 25 nt without affecting silencing activity.While not wishing to be bound by any particular theory, it is believedthat these shRNAs resemble the double-stranded RNA (dsRNA) products ofthe DICER RNase and, in any event, have the same capacity for inhibitingexpression of a specific gene. The shRNA can be expressed from alentiviral vector. An miRNA is a single-stranded RNA of about 10 to 70nucleotides in length that are initially transcribed as pre-miRNAcharacterized by a “stem-loop” structure and which are subsequentlyprocessed into mature miRNA after further processing through the RISC.

Molecules that mediate RNAi, including without limitation siRNA, can beproduced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199,2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase(Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc NatlAcad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-strandedRNA using a nuclease such as E. coli RNase III (Yang et al., Proc NatlAcad Sci USA 99:9942-9947, 2002).

According to another aspect, the disclosure provides polynucleotideantagonists including but not limited to, a decoy DNA, a double-strandedDNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, aviral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA,a double-stranded RNA, a molecule capable of generating RNAinterference, or combinations thereof.

In some embodiments, the polynucleotide antagonists of the disclosureare aptamers. Aptamers are nucleic acid molecules, includingdouble-stranded DNA and single-stranded RNA molecules, which bind to andform tertiary structures that specifically bind to a target molecule,such as a GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6,BMP7, Nodal, ActRIIA, and ActRIIB polypeptide. The generation andtherapeutic use of aptamers are well established in the art. See, e.g.,U.S. Pat. No. 5,475,096. Additional information on aptamers can be foundin U.S. Patent Application Publication No. 20060148748. Nucleic acidaptamers are selected using methods known in the art, for example viathe Systematic Evolution of Ligands by Exponential Enrichment (SELEX)process. SELEX is a method for the in vitro evolution of nucleic acidmolecules with highly specific binding to target molecules as describedin, e.g., U.S. Pat. Nos. 5,475,096, 5,580,737, 5,567,588, 5,707,796,5,763,177, 6,011,577, and 6,699,843. Another screening method toidentify aptamers is described in U.S. Pat. No. 5,270,163. The SELEXprocess is based on the capacity of nucleic acids for forming a varietyof two- and three-dimensional structures, as well as the chemicalversatility available within the nucleotide monomers to act as ligands(form specific binding pairs) with virtually any chemical compound,whether monomeric or polymeric, including other nucleic acid moleculesand polypeptides. Molecules of any size or composition can serve astargets. The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievedesired binding affinity and selectivity. Starting from a mixture ofnucleic acids, which can comprise a segment of randomized sequence, theSELEX method includes steps of contacting the mixture with the targetunder conditions favorable for binding; partitioning unbound nucleicacids from those nucleic acids which have bound specifically to targetmolecules; dissociating the nucleic acid-target complexes; amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand enriched mixture of nucleic acids. The steps of binding,partitioning, dissociating and amplifying are repeated through as manycycles as desired to yield highly specific high affinity nucleic acidligands to the target molecule.

Typically, such binding molecules are separately administered to theanimal [see, e.g., O'Connor (1991) J. Neurochem. 56:560], but suchbinding molecules can also be expressed in vivo from polynucleotidestaken up by a host cell and expressed in vivo [see, e.g.,Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)].

Any of the polynucleotide ActRII antagonists disclosed herein (e.g., apolynucleotide antagonist of one or more of GDF11, GDF8, activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and/or ActRIIB) can be combined with one or more additional ActRIIantagonist agents of the disclosure to achieve the desired effect (e.g.,increase red blood cell levels and/or hemoglobin in a subject in needthereof, treat or prevent an anemia, treat MDS or sideroblastic anemias,treat or prevent one or more complications of MDS or sideroblasticanemias). For example, an polynucleotide ActRII antagonist disclosedherein (e.g., a polynucleotide antagonist of one or more of GDF11, GDF8,activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7,Nodal, ActRIIA, and/or ActRIIB) can be used in combination with i) oneor more additional polynucleotide ActRII antagonists disclosed herein,ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIAand/or ActRIIB polypeptides), iii) one or more GDF traps disclosedherein; iv) one or more ActRII antagonist antibodies disclosed herein(e.g., an anti-GDF11 antibody, an anti-activin B antibody, ananti-activin C antibody, an anti-activin E antibody, an anti-GDF11antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody); v) oneor more small molecule ActRII antagonists disclosed herein (e.g., asmall molecule antagonist of one or more of GDF11, GDF8, activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and/or ActRIIB); vi) one or more follistatin polypeptides disclosedherein; and/or vii) one or more FLRG polypeptides disclosed herein.

F. Other Antagonists

In other aspects, an agent for use in accordance with the methodsdisclosed herein is a follistatin polypeptide, which may be used aloneor in combination with one or more erythropoiesis stimulating agents(e.g., EPO) or other supportive therapies [e.g., hematopoietic growthfactors (e.g., G-CSF or GM-CSF), transfusion of red blood cells or wholeblood, iron chelation therapy], to, e.g., increase red blood cell levelsin a subject in need thereof, treat or prevent an anemia in a subject inneed thereof (including, e.g., reduction of transfusion burden), treatMDS or sideroblastic anemias in a subject in need thereof, and/or treator prevent one or more complications of MDS or sideroblastic anemias(e.g., anemia, blood transfusion requirement, neutropenia, ironoverload, acute myocardial infarction, hepatic failure, hepatomegaly,splenomegaly, progression to acute myeloid lymphoma) and or treat orprevent a disorder associated with SF3B1, DNMT3A, and/or TET2 mutationsin a subject in need thereof. The term “follistatin polypeptide”includes polypeptides comprising any naturally occurring polypeptide offollistatin as well as any variants thereof (including mutants,fragments, fusions, and peptidomimetic forms) that retain a usefulactivity, and further includes any functional monomer or multimer offollistatin. In certain preferred embodiments, follistatin polypeptidesof the disclosure bind to and/or inhibit activin activity, particularlyactivin A (e.g., activin-mediated activation of ActRIIA and/or ActRIIBSMAD 2/3 signaling). Variants of follistatin polypeptides that retainactivin binding properties can be identified based on previous studiesinvolving follistatin and activin interactions. For example,WO2008/030367 discloses specific follistatin domains (“FSDs”) that areshown to be important for activin binding. As shown below in SEQ ID NOs:18-20, the follistatin N-terminal domain (“FSND” SEQ ID NO:18), FSD2(SEQ ID NO: 20), and to a lesser extent FSD1 (SEQ ID NO: 19) representexemplary domains within follistatin that are important for activinbinding. In addition, methods for making and testing libraries ofpolypeptides are described above in the context of ActRII polypeptides,and such methods also pertain to making and testing variants offollistatin. Follistatin polypeptides include polypeptides derived fromthe sequence of any known follistatin having a sequence at least about80% identical to the sequence of a follistatin polypeptide, andoptionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greateridentity. Examples of follistatin polypeptides include the maturefollistatin polypeptide or shorter isoforms or other variants of thehuman follistatin precursor polypeptide (SEQ ID NO: 16) as described,for example, in WO2005/025601.

The human follistatin precursor polypeptide isoform FST344 is asfollows:

(SEQ ID NO: 16; NCBI Reference No. NP_037541.1) 1mvrarhqpgg lcllllllcq fmedrsaqag ncwlrqakng rcqvlyktel 51 skeeccstgrlstswteedv ndntlfkwmi fnggapncip cketcenvdc 101 gpgkkcrmnk knkprcvcapdcsnitwkgp vcgldgktyr necallkarc 151 keqpelevqy qgrckktcrd vfcpgsstcvvdqtnnaycv tcnricpepa 201 sseqylcgnd gvtyssachl rkatcllgrs iglayegkcikakscediqc 251 tggkkclwdf kvgrgrcslc delcpdsksd epvcasdnat yasecamkea301 acssgvllev khsgscnsis edteeeeede dqdysfpiss ilew

The signal peptide is underlined; also underlined above are the last 27residues which represent the C-terminal extension distinguishing thisfollistatin isoform from the shorter follistatin isoform FST317 shownbelow.

The human follistatin precursor polypeptide isoform FST317 is asfollows:

(SEQ ID NO: 17; NCBI Reference No. NP_006341.1) 1MVRARHQPGG LCLLLLLLCQ FMEDRSAQAG NCWLRQAKNG RCQVLYKTEL 51 SKEECCSTGRLSTSWTEEDV NDNTLFKWMI FNGGAPNCIP CKETCENVDC 101 GPGKKCRMNK KNKPRCVCAPDCSNITWKGP VCGLDGKTYR NECALLKARC 151 KEQPELEVQY QGRCKKTCRD VFCPGSSTCVVDQTNNAYCV TCNRICPEPA 201 SSEQYLCGND GVTYSSACHL RKATCLLGRS IGLAYEGKCIKAKSCEDIQC 251 TGGKKCLWDF KVGRGRCSLC DELCPDSKSD EPVCASDNAT YASECAMKEA301 ACSSGVLLEV KHSGSCNThe signal peptide is underlined.

The follistatin N-terminal domain (FSND) sequence is as follows:

(SEQ ID NO: 18; FSND) GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCK

The FSD1 and FSD2 sequences are as follows:

(SEQ ID NO: 19; FSD1) ETCENVDCGPGKKCRMNKKNKPRCV (SEQ ID NO: 20; FSD2)KTCRDVFCPGSSTCVVDQTNNAYCVT

In other aspects, an agent for use in accordance with the methodsdisclosed herein is a follistatin-like related gene (FLRG), also knownas follistatin-related protein 3 (FSTL3). The term “FLRG polypeptide”includes polypeptides comprising any naturally occurring polypeptide ofFLRG as well as any variants thereof (including mutants, fragments,fusions, and peptidomimetic forms) that retain a useful activity. Incertain preferred embodiments, FLRG polypeptides of the disclosure bindto and/or inhibit activin activity, particularly activin A (e.g.,activin-mediated activation of ActRIIA and/or ActRIIB SMAD 2/3signaling). Variants of FLRG polypeptides that retain activin bindingproperties can be identified using routine methods to assay FLRG andactivin interactions (see, e.g., U.S. Pat. No. 6,537,966). In addition,methods for making and testing libraries of polypeptides are describedabove in the context of ActRII polypeptides and such methods alsopertain to making and testing variants of FLRG. FLRG polypeptidesinclude polypeptides derived from the sequence of any known FLRG havinga sequence at least about 80% identical to the sequence of an FLRGpolypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greateridentity.

The human FLRG precursor (follistatin-related protein 3 precursor)polypeptide is as follows:

(SEQ ID NO: 21; NCBI Reference No. NP_005851.1) 1MRPGAPGPLW PLPWGALAWA VGFVSSMGSG NPAPGGVCWL QQGQEATCSL 51 VLQTDVTRAECCASGNIDTA WSNLTHPGNK INLLGFLGLV HCLPCKDSCD 101 GVECGPGKAC RMLGGRPRCECAPDCSGLPA RLQVCGSDGA TYRDECELRA 151 ARCRGHPDLS VMYRGRCRKS CEHVVCPRPQSCVVDQTGSA HCVVCRAAPC 201 PVPSSPGQEL CGNNNVTYIS SCHMRQATCF LGRSIGVRHAGSCAGTPEEP 251 PGGESAEEEE NFVThe signal peptide is underlined.

In certain embodiments, functional variants or modified forms of thefollistatin polypeptides and FLRG polypeptides include fusion proteinshaving at least a portion of the follistatin polypeptide or FLRGpolypeptide and one or more fusion domains, such as, for example,domains that facilitate isolation, detection, stabilization ormultimerization of the polypeptide. Suitable fusion domains arediscussed in detail above with reference to the ActRII polypeptides. Insome embodiment, an antagonist agent of the disclosure is a fusionprotein comprising an activin-binding portion of a follistatinpolypeptide fused to an Fc domain. In another embodiment, an antagonistagent of the disclosure is a fusion protein comprising an activinbinding portion of an FLRG polypeptide fused to an Fc domain.

Any of the follistatin polypeptides disclosed herein may be combinedwith one or more additional ActRII antagonist agents of the disclosureto achieve the desired effect (e.g., increase red blood cell levelsand/or hemoglobin in a subject in need thereof, treat or prevent ananemia, treat MDS or sideroblastic anemias, treat or prevent one or morecomplications of MDS or sideroblastic anemias). For example, afollistatin polypeptide disclosed herein can be used in combination withi) one or more additional follistatin polypeptides disclosed herein, ii)one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/orActRIIB polypeptides), iii) one or more GDF traps disclosed herein; iv)one or more ActRII antagonist antibodies disclosed herein (e.g., ananti-GDF11 antibody, an anti-activin B antibody, an anti-activin Cantibody, an anti-activin E antibody, an anti-GDF11 antibody, ananti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7 antibody, ananti-ActRIIA antibody, or an anti-ActRIIB antibody); v) one or moresmall molecule ActRII antagonists disclosed herein (e.g., a smallmolecule antagonist of one or more of GDF11, GDF8, activin A, activin B,activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/orActRIIB); vi) one or more polynucleotide ActRII antagonists disclosedherein (e.g., a polynucleotide antagonist of one or more of GDF11, GDF8,activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7,Nodal, ActRIIA, and/or ActRIIB); and/or one or more FLRG polypeptidesdisclosed herein.

Similarly, any of the FLRG polypeptides disclosed herein may be combinedwith one or more additional ActRII antagonist agents of the disclosureto achieve the desired effect. For example, a FLRG polypeptide disclosedherein can be used in combination with i) one or more additional FLRGpolypeptides disclosed herein, ii) one or more ActRII polypeptidesdisclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) oneor more GDF traps disclosed herein; iv) one or more ActRII antagonistantibodies disclosed herein (e.g., an anti-GDF11 antibody, ananti-activin B antibody, an anti-activin C antibody, an anti-activin Eantibody, an anti-GDF11 antibody, an anti-GDF8 antibody, an anti-BMP6antibody, an-anti-BMP7 antibody, an anti-ActRIIA antibody, or ananti-ActRIIB antibody); v) one or more small molecule ActRII antagonistsdisclosed herein (e.g., a small molecule antagonist of one or more ofGDF11, GDF8, activin A, activin B, activin AB, activin C, activin E,BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); vi) one or morepolynucleotide ActRII antagonists disclosed herein (e.g., apolynucleotide antagonist of one or more of GDF11, GDF8, activin A,activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA,and/or ActRIIB); and/or one or more follistatin polypeptides disclosedherein.

3. Screening Assays

In certain aspects, the present disclosure relates to the use of thesubject ActRII polypeptides (e.g., ActRIIA and ActRIIB polypeptides) andGDF trap polypeptides to identify compounds (agents) which are agonistor antagonists of ActRIIB polypeptides. Compounds identified throughthis screening can be tested to assess their ability to modulate redblood cell, hemoglobin, and/or reticulocyte levels in vivo or in vitro.These compounds can be tested, for example, in animal models.

There are numerous approaches to screening for therapeutic agents forincreasing red blood cell or hemoglobin levels by targeting ActRIIsignaling (e.g., ActRIIA and/or ActRIIB SMAD 2/3 and/or SMAD 1/5/8signaling). In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturbActRII-mediated effects on a selected cell line. In certain embodiments,the assay is carried out to screen and identify compounds thatspecifically inhibit or reduce binding of an ActRII polypeptide or GDFtrap polypeptide to its binding partner, such as an ActRII ligand (e.g.,activin A, activin B, activin AB, activin C, Nodal, GDF8, GDF11 orBMP7). Alternatively, the assay can be used to identify compounds thatenhance binding of an ActRII polypeptide or GDF trap polypeptide to itsbinding partner such as an ActRII ligand. In a further embodiment, thecompounds can be identified by their ability to interact with an ActRIIpolypeptide or GDF trap polypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. Incertain embodiments, the test agent is a small organic molecule having amolecular weight of less than about 2,000 Daltons.

The test compounds of the disclosure can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S-transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug-screening programs which test libraries of compounds andnatural extracts, high-throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIpolypeptide or a GDF trap polypeptide and its binding partner (e.g., anActRII ligand).

Merely to illustrate, in an exemplary screening assay of the presentdisclosure, the compound of interest is contacted with an isolated andpurified ActRIIB polypeptide which is ordinarily capable of binding toan ActRIIB ligand, as appropriate for the intention of the assay. To themixture of the compound and ActRIIB polypeptide is then added to acomposition containing an ActRIIB ligand (e.g., GDF11). Detection andquantification of ActRIIB/ActRIIB ligand complexes provides a means fordetermining the compound's efficacy at inhibiting (or potentiating)complex formation between the ActRIIB polypeptide and its bindingprotein. The efficacy of the compound can be assessed by generatingdose-response curves from data obtained using various concentrations ofthe test compound. Moreover, a control assay can also be performed toprovide a baseline for comparison. For example, in a control assay,isolated and purified ActRIIB ligand is added to a compositioncontaining the ActRIIB polypeptide, and the formation of ActRIIB/ActRIIBligand complex is quantitated in the absence of the test compound. Itwill be understood that, in general, the order in which the reactantsmay be admixed can be varied, and can be admixed simultaneously.Moreover, in place of purified proteins, cellular extracts and lysatesmay be used to render a suitable cell-free assay system.

Complex formation between an ActRII polypeptide or GDF trap polypeptideand its binding protein may be detected by a variety of techniques. Forinstance, modulation of the formation of complexes can be quantitatedusing, for example, detectably labeled proteins such as radiolabeled(e.g., ³²P, ³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), orenzymatically labeled ActRII polypeptide or GDF trap polypeptide and/orits binding protein, by immunoassay, or by chromatographic detection.

In certain embodiments, the present disclosure contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRII polypeptide of GDF trappolypeptide and its binding protein. Further, other modes of detection,such as those based on optical waveguides (see, e.g., PCT Publication WO96/26432 and U.S. Pat. No. 5,677,196), surface plasmon resonance (SPR),surface charge sensors, and surface force sensors, are compatible withmany embodiments of the disclosure.

Moreover, the present disclosure contemplates the use of an interactiontrap assay, also known as the “two-hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRII polypeptide orGDF trap polypeptide and its binding partner. See, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) JBiol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). In aspecific embodiment, the present disclosure contemplates the use ofreverse two-hybrid systems to identify compounds (e.g., small moleculesor peptides) that dissociate interactions between an ActRII polypeptideor GDF trap and its binding protein [see, e.g., Vidal and Legrain,(1999) Nucleic Acids Res 27:919-29; Vidal and Legrain, (1999) TrendsBiotechnol 17:374-81; and U.S. Pat. Nos. 5,525,490; 5,955,280; and5,965,368].

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRII polypeptide or GDF trap polypeptide.The interaction between the compound and the ActRII polypeptide or GDFtrap polypeptide may be covalent or non-covalent. For example, suchinteraction can be identified at the protein level using in vitrobiochemical methods, including photo-crosslinking, radiolabeled ligandbinding, and affinity chromatography [see, e.g., Jakoby W B et al.(1974) Methods in Enzymology 46:1]. In certain cases, the compounds maybe screened in a mechanism-based assay, such as an assay to detectcompounds which bind to an ActRII polypeptide of GDF trap polypeptide.This may include a solid-phase or fluid-phase binding event.Alternatively, the gene encoding an ActRII polypeptide or GDF trappolypeptide can be transfected with a reporter system (e.g.,β-galactosidase, luciferase, or green fluorescent protein) into a celland screened against the library preferably by high-throughput screeningor with individual members of the library. Other mechanism-based bindingassays may be used; for example, binding assays which detect changes infree energy. Binding assays can be performed with the target fixed to awell, bead or chip or captured by an immobilized antibody or resolved bycapillary electrophoresis. The bound compounds may be detected usuallyusing colorimetric endpoints or fluorescence or surface plasmonresonance.

4. Exemplary Therapeutic Uses

In certain aspects, the disclosure provides methods of treating MDS andsideroblastic anemias, particularly treating or preventing one or moresubtypes or complications of MDS, with one or more ActRII antagonists,including the treatment of patients with MDS characterized by thepresence of ring sideroblasts and/or one or more mutations in the SF3B1,DNMT3A, and/or TET2 genes. In particular, the disclosure providesmethods for using an ActRII antagonist, or combination of ActRIIantagonists, to treat or prevent one or more complications of MDS andsideroblastic anemias including, for example, anemia, neutropenia,splenomegaly, blood transfusion requirement, development of acutemyeloid leukemia, iron overload, and complications of iron overload,among which are congestive heart failure, cardiac arrhythmia, myocardialinfarction, other forms of cardiac disease, diabetes mellitus, dyspnea,hepatic disease, and adverse effects of iron chelation therapy.

In particular, the disclosure provides methods for using an ActRIIantagonist, or combination of ActRII antagonists, to treat or preventanemia or other complications in a subtype of MDS, including MDSpatients with elevated numbers of erythroblasts (hypercellularity) inbone marrow; in MDS patients with more than 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%sideroblasts in bone marrow; in MDS patients with refractory anemia withring sideroblasts (RARS); in MDS patients with refractory anemia withring sideroblasts and thrombocytosis (RARS-T); in MDS patients withrefractory cytopenia with unilineage dysplasia (RCUD); in MDS patientswith refractory cytopenia with multilineage dysplasia and ringsideroblasts (RCMD-RS); in MDS patients with a somatic mutation inSF3B1, SRSF2, DNMT3A, or TET2; in MDS patients without a somaticmutation in ASXL1 or ZRSR2; in MDS patients with iron overload; and inMDS patients with neutropenia.

Also in particular, the disclosure provides methods for using an ActRIIantagonist, or combination of ActRII antagonists, to treat or preventanemia or other complications of a sideroblastic anemia, including butnot limited to refractory anemia with ring sideroblasts (RARS);refractory anemia with ring sideroblasts and thrombocytosis (RARS-T);refractory cytopenia with multilineage dysplasia and ring sideroblasts(RCMD-RS); sideroblastic anemia associated with alcoholism; drug-inducedsideroblastic anemia; sideroblastic anemia resulting from copperdeficiency (zinc toxicity); sideroblastic anemia resulting fromhypothermia; X-linked sideroblastic anemia (XLSA); SLC25A38 deficiency;glutaredoxin 5 deficiency; erythropoietic protoporphyria; X-linkedsideroblastic anemia with ataxia (XLSA/A); sideroblastic anemia withB-cell immunodeficiency, fevers, and developmental delay (SIFD); Pearsonmarrow-pancreas syndrome; myopathy, lactic acidosis, and sideroblasticanemia (MLASA); thiamine-responsive megaloblastic anemia (TRIVIA); andsyndromic/nonsyndromic sideroblastic anemia of unknown cause.

In certain aspects the disclosure provides methods for treating orpreventing disorders or complications of a disorder that is associatedwith germ line or somatic mutations in SF3B1, DNMT3A, and/or TET2, suchas myelodysplastic syndrome, chronic lymphocytic leukemia (CLL), andacute myeloid leukemia (AML) as well as in breast cancer, pancreaticcancer, gastric cancer, prostate cancer, and uveal melanoma. In certainaspects the disorder may be in a subject that has bone marrow cells thattest positive for an SF3B1, DNMT3A, and/or TET2 mutation, particularlymyelodysplastic syndrome, CLL and AML. Optionally a mutation in theSF3B1 gene is in an exon, intron or 5′ or 3′ untranslated region.Optionally a mutation in SF3B1, DNMT3A, and/or TET2 causes a change inthe amino acid sequence or does not cause a change in the amino acidsequence of the protein encoded by the gene. Optionally a mutation inthe SF3B1 gene causes a change in the amino acid of the protein encodedby the gene selected from the following changes: K182E, E491G, R590K,E592K, R625C, R625G, N626D, N626S, H662Y, T663A, K666M, K666Q, K666R,Q670E, G676D, V701I, I1704N, 1704V, G740R, A744P, D781G, A1188V, N619K,N626H, N626Y, R630S, 1704T, G740E, K741N, G742D, D894G, Q903R, R1041H,I1241T, G347V, E622D, Y623C, R625H, R625L, H662D, H662Q, T663I, K666E,K666N, K666T, K700E, and V701F. Optionally a mutation in the DNMT3A genecauses a change in the amino acid of the protein encoded by the geneselected from the following changes: R882C, R882H, P904L, and P905P.Optionally a mutation in the DNMT3A gene introduces a premature stopcodon. For example, in some embodiments, a mutation in the DNMT3A genethat introduces a premature stop codon is selected from the followingpositions: Y436X and W893X. Optionally a mutation in the TET2 genecauses a change in the amino acid of the protein encoded by the geneselected from the following changes: E47Q, Q1274R, W1291R, G1370R,N1387S, and Y1724H. Optionally a mutation in the TET2 gene introduces apremature stop codon. For example, in some embodiments, a mutation inthe TET2 gene that introduces a premature stop codon is selected fromthe following positions: R550X, Q1009X, Y1337X, R1404X, R1516X, andQ1652X.

The terms “subject,” an “individual,” or a “patient” are interchangeablethroughout the specification and generally refer to mammals. Mammalsinclude, but are not limited to, domesticated animals (e.g., cows,sheep, cats, dogs, and horses), primates (e.g., humans and non-humanprimates such as monkeys), rabbits, and rodents (e.g., mice and rats).

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The term “treating” as used herein includes amelioration or eliminationof the condition once it has been established. In either case,prevention or treatment may be discerned in the diagnosis provided by aphysician or other health care provider and the intended result ofadministration of the therapeutic agent.

In general, treatment or prevention of a disease or condition asdescribed in the present disclosure is achieved by administering one ormore of the ActRII antagonists (e.g., an ActRIIA and/or ActRIIBantagonist) of the present disclosure in an effective amount. Aneffective amount of an agent refers to an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. A “therapeutically effective amount” of an agent ofthe present disclosure may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theagent to elicit a desired response in the individual. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result.

Myelodysplastic syndromes (MDS) are a diverse collection ofhematological disorders characterized by ineffective production ofmyeloid blood cells and risk of transformation to acute myeloidleukemia. In MDS patients, hematopoietic stem cells do not mature intohealthy red blood cells, white blood cells, or platelets. MDS disordersinclude, for example, refractory anemia, refractory cytopenia withunilineage dysplasia (RCUD), refractory anemia with ringed sideroblasts(RARS), refractory anemia with ringed sideroblasts associated withmarked thrombocytosis (RARS-T), refractory anemia with excess blasts(RAEB-1), refractory anemia with excess blasts in transformation(RAEB-2), refractory cytopenia with multilineage dysplasia (RCMD), MDSunclassified (MDS-U), and myelodysplastic syndrome associated with anisolated 5q chromosome abnormality [MDS with del(5q)].

Allogenic stem-cell transplantation is the only known potentiallycurative therapy for MDS. However, only a minority of patients undergothis procedure due to advanced age, medical comorbidities, and limitedavailability of appropriate stem cell donors. Even for those patientswho proceed to allogenic stem-cell transplantation, significanttreatment-related mortality and morbidity, including acute and chronicgraft-versus-host-disease, and high relapse rates compromise long-termdisease-free survival [Zeidan et al. (2013) Blood Rev 27:243-259]. Forthese reasons, the majority of patients with MDS are still managed on anon-curative intent therapeutic paradigm. Treatment is based onprognostic factors that predict survival or progression to acute myeloidleukemia. Survival of lower-risk MDS patients ranges from several monthsto more than a decade, and most of these patients die from causesdirectly related to complications of MDS [Dayyani et al. (2010) Cancer116:2174-2179]. Therefore, therapeutic strategies for lower-riskpatients are adapted to the specific patient's situation, includingseverity and type of cytopenias and expected survival. Lower-riskpatients have multiple therapeutic options, including treatment withgrowth factors, lenalidomide in the case of del(5q) syndrome,thalidomide, pomalidomide, hypomethylating agents such as azacitidine ordecitibine, and potentially investigational agents. In contrast,higher-risk MDS patients who are not eligible for allogenic stem-celltransplantation are typically treated with hypomethylating agents,intensive chemotherapy, or investigational agents [Garcia-Manero et al.(2011) 29:516-523].

Since MDS manifest as irreversible defects in both quantity and qualityof hematopoietic cells, most MDS patients are afflicted with chronicanemia. Approximately 80% to 90% of MDS patients develop anemia duringthe course of their disease, of whom at least 40% become RBCtransfusion-dependent [Santini (2011) Oncologist 16:35-42; Malcovati etal. (2005) J Clin Oncol 23:7594-7603; Leitch (2011) Blood Rev 25:17-31].Patients in higher-risk MDS groups (according to IPSS classification)are even more likely to become transfusion-dependent; for example, inone study 79% of high-risk category patients versus 39% of low-riskpatients required chronic transfusions to treat or prevent severe anemia[Oscan et al. (2013) Expert Rev Hematol 6:165-189]. Low hemoglobinlevels result in poor oxygenation of the brain and peripheral organs; asa result, MDS patients typically suffer from lethargy, decreased mentalalertness, physical weakness, and poor concentration. These symptoms arelinked to a reduced health-related quality of life. In addition,hemoglobin thresholds are independently associated with significantmorbidity and mortality in MDS. In female and male patients withhemoglobin levels lower than 8 g/dL and 9 g/dL, respectively, the riskof morbidity and mortality increases, mainly due to an increased risk ofcardiac complications [Malcovati et al. (2011) Haematologica96:1433-1440]. Low hemoglobin levels and dependence on red blood celltransfusions have been associated with inferior cardiovascular outcomesand increased mortality in patients with MDS, representing a strongrationale for aggressive management of anemia in MDS [Goldberg et al.(2010) J Clin Oncol 28:2847-2852; Leitch (2011) Blood Rev 25:17-31;Oliva et al. (2011) Am J Blood Res 1:160-166; Ozcan et al. (2013) ExpertRev Hematol 6:165-189].

MDS patients eventually require blood transfusions and/or treatment witherythropoietic growth factors (e.g., ESAs such as EPO) alone or incombination with a colony-stimulating factor [e.g., an analog ofgranulocyte colony-stimulating factor (G-CSF) such as filgrastim or ananalog of granulocyte macrophage colony-stimulating factor (GM-GSF) suchas sargramostim] to increase red blood cell levels. The frequency oftransfusions depends on the extent of the disease and on the presence ofcomorbidities. Chronic transfusions are known to increase hemoglobinlevels, which in turn improve brain and peripheral tissue oxygenation,thereby improving physical activity and mental alertness. However, manyMDS patients develop side-effects from the use of such therapies. Forexample, patients who receive frequent red blood cell transfusions candevelop tissue and organ damage from iron accumulation and generation oftoxic reactive oxygen species. Accordingly, one or more ActRIIantagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, anActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc.),optionally combined with an EPO receptor activator, may be used to treatpatients with MDS or sideroblastic anemias. In certain embodiments,patients suffering from MDS or a siderblastic anemia may be treatedusing one or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.), optionally in combination with an EPO receptoractivator. In other embodiments, patients suffering from MDS or asideroblastic anemia may be treated using a combination of one or moreActRII antagonist agents of the disclosure (e.g., a GDF-ActRIIantagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap,etc.) and one or more additional therapeutic agents for treating MDSincluding, for example, ESAs including, e.g., epoetin alfa, epoetin beta(e.g., NeoRecormon), epoetin delta (e.g., Dynepo), epoetin omega,darbepoetin alfa (e.g., Aranesp), methoxy-polyethylene-glycol epoetinbeta (e.g., Micera), and synthetic erythropoiesis protein (SEP); G-CSFanalogs, including filgrastim; GM-CSF analogs, including sargramostim;lenalidomide; thalidomide; pomalidomide, hypomethylating agents,including azacitidine and decitabine; iron-chelating agents, includingdeferoxamine (a.k.a., desferrioxamine B, desferoxamine B, DFO-B, DFOA,DFB, or desferal), deferiprone (a.k.a., Ferriprox), and deferasirox(a.k.a., bis-hydroxyphenyl-triazole, ICL670, or Exjade™); thrombopoietinmimetics, including romiplostim and eltrombopag; chemotherapeuticagents, including cytarabine (ara-C) alone or in combination withidarubicin, topotecan, or fludarabine; immunosuppressants, includingantithymocyte globulin, alemtuzumab, and cyclosporine; histonedeacetylase inhibitors (HDAC inhibitors), including vorinostat, valproicacid, phenylbutyrate, entinostat, MGCD0103, and other class I nuclearHDAC inhibitors, class II non-nuclear HDAC inhibitors, pan HDACinhibitors, and isoform-specific HDAC inhibitors; farnesyltransferaseinhibitors, including as tipifarnib and lonafarnib; tumor necrosisfactor-alpha (TNF-α) inhibitors, including etanercept or infliximab;inhibitors of glutathione-S-transferase (GST) P1-1, includingezatiostat; and inhibitors of CD33, including gemtuzumab ozogamicin.

One or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.), optionally combined with an EPO receptor activatorand/or one or more additional therapies, may be used to increase redblood cell levels, hemoglobin levels, and/or hematocrit levels in apatient with anemia (MDS or sideroblastic anemia). When monitoringhemoglobin and/or hematocrit levels in humans, a level of less thannormal for the appropriate age and gender category may be indicative ofanemia, although individual variations are taken into account. Forexample, a hemoglobin level from 10-12.5 g/dl, and typically about 11.0g/dl is considered to be within the normal range in healthy adults,although, in terms of therapy, a lower target level may cause fewercardiovascular side effects. See, e.g., Jacobs et al. (2000) NephrolDial Transplant 15, 15-19. Alternatively, hematocrit levels (percentageof the volume of a blood sample occupied by the cells) can be used as ameasure of anemia. Hematocrit levels for healthy individuals range fromabout 41-51% for adult males and from 35-45% for adult females. Incertain embodiments, a patient may be treated with a dosing regimenintended to restore the patient to a target level of red blood cells,hemoglobin, and/or hematocrit or allow the reduction or elimination ofred blood cell transfusions while maintaining an acceptable level of redblood cells, hemoglobin and/or hematocrit. As hemoglobin and hematocritlevels vary from person to person, optimally, the target hemoglobinand/or hematocrit level can be individualized for each patient.

Neutropenia denotes a condition of abnormally low levels of circulatinggranulocytes and is found in approximately 40% of MDS patients [Steensmaet al. (2006) Mayo Clin Proc 81:104-130]. Common complaints of patientswith neutropenia include fatigue and frequent bacterial infections,especially of the skin. However, neutropenia can also result in seriouscomplications in patients with MDS, and infection is the most commoncause of MDS-associated death. Treatment with granulocytecolony-stimulating factor (filgrastim) can help keep neutrophil countsabove 1×10⁹/L for severely neutropenic patients [Akhtari (2011) Oncology(Williston Park) 25:480-486], but myeloid growth factors do not clearlymodify disease history and may only increase production of functionallydefective neutrophils lacking bactericidal capacity [Dayyani et al.(2010) Cancer 116:2174-2179; Steensma (2011) Semin Oncol 38:635-647].Prophylactic antibiotics have no proven role in patients with MDS andare not recommended for patients with neutropenia related to MDS.However, neutropenic fever in MDS patients should be regarded as amedical emergency, requiring immediate administration of empiricbroad-spectrum antibiotics and often hospitalization [Barzi et al.(2010) Cleve Clin J Med 77:37-44]. In some embodiments, one or moreActRII antagonist agents of the disclosure (e.g., a GDF-ActRIIantagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap,etc.), optionally combined with an EPO receptor activator and/or one ormore additional therapies such as a G-CSF or GM-CSF therapy, may be usedto treat neutropenia in MDS patients. One or more ActRII antagonistagents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIApolypeptide, an ActRIIB polypeptide, a GDF trap, etc.), optionallycombined with an EPO receptor activator and/or one or more additionaltherapies, may be used to reduce the frequency of granulocytetransfusions in MDS patients.

Patients with MDS or sideroblastic anemia who receive frequenttransfusions of red blood cells or whole blood are prone to developtransfusional iron overload, which may partly explain why transfusiondependency in MDS is associated with reduced likelihood of survival.Nevertheless, the use of iron chelation therapy in transfusion-dependentMDS patients remains controversial, because retrospective and registrydata suggest chelated patients may live longer than unchelated patients,yet there are no prospective randomized trial data demonstrating amorbidity or mortality benefit from chelation, and currently approvedagents are inconvenient (deferroxamine) or costly and poorly toleratedby many patients (deferasirox) [Steensma et al. (2013) Best Pract ResClin Haematol 26:431-444; Lyons et al. (2014) Leuk Res 38:149-154].

In some embodiments, one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.), optionally combined with an EPOreceptor activator and/or one or more additional therapies, may be usedto prevent or reverse complications of iron overload in patients withMDS or sideroblastic anemia. In certain aspects, one or more ActRIIantagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, anActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc.),optionally combined with an EPO receptor activator and/or one or moreadditional therapies, may be used to prevent or reverse a cardiaccomplication of iron overload including, e.g., increased cardiac output,cardiomegaly, cardiomyopathy, left ventricular hypertrophy, acutemyocardial infarction, arrhythmia, and congestive heart failure. Incertain aspects, one or more ActRII antagonist agents of the disclosure(e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIBpolypeptide, a GDF trap, etc.), optionally combined with an EPO receptoractivator and/or one or more additional therapies, may be used to reduceliver iron content and/or prevent or reverse a hepatic complication ofiron overload including, e.g., liver enlargement (hepatomegaly), liverfibrosis (increase in scar tissue), and cirrhosis (extensive scarring).In certain aspects, one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.), optionally combined with an EPOreceptor activator and/or one or more additional therapies, may be usedto prevent or reverse an endocrine complication of iron overloadincluding, e.g., diabetes mellitus.

In certain aspects, ActRII antagonist agents of the disclosure may beadministered to a subject in need thereof in combination with one ormore additional agents [for example, ESAs; G-CSF analogs, includingfilgrastim; GM-CSF analogs, including sargramostim; lenalidomide;thalidomide; pomalidomide, hypomethylating agents, including azacitidineand decitabine; iron-chelating agents, including deferoxamine anddeferasirox; thrombopoietin mimetics, including romiplostim andeltrombopag; chemotherapeutic agents, including cytarabine (ara-C) aloneor in combination with idarubicin, topotecan, or fludarabine;immunosuppressants, including antithymocyte globulin, alemtuzumab, andcyclosporine; histone deacetylase inhibitors (HDAC inhibitors),including vorinostat, valproic acid, phenylbutyrate, entinostat,MGCD0103, and other class I nuclear HDAC inhibitors, class IInon-nuclear HDAC inhibitors, pan HDAC inhibitors, and isoform-specificHDAC inhibitors; farnesyltransferase inhibitors, including as tipifarniband lonafarnib; tumor necrosis factor-alpha (TNF-α) inhibitors,including etanercept or infliximab; inhibitors ofglutathione-S-transferase (GST) P1-1, including ezatiostat; andinhibitors of CD33, including gemtuzumab ozogamicin.] or supportivetherapies [e.g., red blood cell transfusion, granulocyte transfusion,thrombocyte (platelet) transfusion] for treating MDS and sideroblasticanemia or one or more complications of MDS and sideroblastic anemia.

As used herein, “in combination with” or “conjoint administration”refers to any form of administration such that additional therapies(e.g., second, third, fourth, etc.) are still effective in the body(e.g., multiple compounds are simultaneously effective in the patient,which may include synergistic effects of those compounds). Effectivenessmay not correlate to measurable concentration of the agent in blood,serum, or plasma. For example, the different therapeutic compounds canbe administered either in the same formulation or in separateformulations, either concomitantly or sequentially, and on differentschedules. Thus, an individual who receives such treatment can benefitfrom a combined effect of different therapies. One or more GDF11 and/oractivin B antagonist agents (optionally further antagonists of one ormore of GDF8, activin A, activin C, activin E, and BMP6) of thedisclosure can be administered concurrently with, prior to, orsubsequent to, one or more other additional agents or supportivetherapies. In general, each therapeutic agent will be administered at adose and/or on a time schedule determined for that particular agent. Theparticular combination to employ in a regimen will take into accountcompatibility of the antagonist of the present disclosure with thetherapy and/or the desired therapeutic effect to be achieved.

In certain embodiments, one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, antibody etc.), optionally combinedwith an EPO receptor activator and/or one or more additional therapies,may be used in combination with transfusion of either red blood cells orwhole blood to treat anemia in patients with MDS or sideroblasticanemias. In patients who receive frequent transfusions of whole blood orred blood cells, normal mechanisms of iron homeostasis can beoverwhelmed, eventually leading to toxic and potentially fatalaccumulation of iron in vital tissues such as heart, liver, andendocrine glands. Regular red blood cell transfusions require exposureto various donor units of blood and hence a higher risk ofalloimmunization. Difficulties with vascular access, availability of andcompliance with iron chelation, and high cost are some of the reasonswhy it can be beneficial to limit the number of red blood celltransfusions. In some embodiments, the methods of the present disclosurerelate to treating MDS or sideroblastic anemia in a subject in needthereof by administering a combination of an ActRII antagonist of thedisclosure and one or more blood cell transfusions. In some embodiments,the methods of the present disclosure relate to treating or preventingone or more complications of MDS or sideroblastic anemia in a subject inneed thereof by administering a combination of an ActRII antagonist ofthe disclosure and one or more red blood cell transfusions. In someembodiments, treatment with one or more ActRII antagonists of thedisclosure is effective at decreasing the transfusion requirement in apatient with MDS or sideroblastic anemia, e.g., reduces the frequencyand/or amount of blood transfusion required to effectively treat MDS orsideroblastic anemia or one or more their complications.

In certain embodiments, one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.), optionally combined with an EPOreceptor activator and/or one or more additional therapies, may be usedin combination with one or more iron-chelating molecules to promote ironexcretion in the urine and/or stool and thereby prevent or reversetissue iron overload in patients with MDS or sideroblastic anemias.Effective iron-chelating agents should be able to selectively bind andneutralize ferric iron, the oxidized form of non-transferrin bound ironwhich likely accounts for most iron toxicity through catalyticproduction of hydroxyl radicals and oxidation products [see, e.g.,Esposito et al. (2003) Blood 102:2670-2677]. These agents arestructurally diverse, but all possess oxygen or nitrogen donor atomsable to form neutralizing octahedral coordination complexes withindividual iron atoms in stoichiometries of 1:1 (hexadentate agents),2:1 (tridentate), or 3:1 (bidentate) [Kalinowski et al. (2005) PharmacolRev 57:547-583]. In general, effective iron-chelating agents also arerelatively low molecular weight (e.g., less than 700 daltons), withsolubility in both water and lipids to enable access to affectedtissues. Specific examples of iron-chelating molecules includedeferoxamine, a hexadentate agent of bacterial origin requiring dailyparenteral administration, and the orally active synthetic agentsdeferiprone (bidentate) and deferasirox (tridentate). Combinationtherapy consisting of same-day administration of two iron-chelatingagents shows promise in patients unresponsive to chelation monotherapyand also in overcoming issues of poor patient compliance withdereroxamine alone [Cao et al. (2011) Pediatr Rep 3(2):e17; andGalanello et al. (2010) Ann NY Acad Sci 1202:79-86].

One or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.), optionally combined with an EPO receptor activatorand/or one or more additional therapies, may be used to increase redblood cell levels, hemoglobin levels, and/or hematocrit levels in apatient with MDS or sideroblastic anemia. When observing hemoglobinand/or hematocrit levels in humans, a level of less than normal for theappropriate age and gender category may be indicative of anemia,although individual variations are taken into account. For example, ahemoglobin level from 10-12.5 g/dl, and typically about 11.0 g/dl isconsidered to be within the normal range in healthy adults, although, interms of therapy, a lower target level may cause fewer cardiovascularside effects. See, e.g., Jacobs et al. (2000) Nephrol Dial Transplant15, 15-19. Alternatively, hematocrit levels (percentage of the volume ofa blood sample occupied by the cells) can be used as a measure foranemia. Hematocrit levels for healthy individuals range from about41-51% for adult males and from 35-45% for adult females. In certainembodiments, a patient may be treated with a dosing regimen intended torestore the patient to a target level of red blood cells, hemoglobin,and/or hematocrit. As hemoglobin and hematocrit levels vary from personto person, optimally, the target hemoglobin and/or hematocrit level canbe individualized for each patient.

One or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.) may be used in combination with EPO receptor activatorsand/or one or more additional therapies to treat anemia. The suboptimalerythropoietin response in some patients with MDS is considered onebiologic rationale for treating MDS-related anemia with ESAs [Greenberget al. (2011) J Natl Compr Canc Netw 9:30-56; Santini (2012) SeminHematol 49:295-303]. Despite not being approved by the FDA for use inMDS-associated anemia, ESAs are in wide clinical use and are the mostcommonly used therapy for MDS [Casadevall et al. (2004) Blood104:321-327; Greenberg et al. (2009) Blood 114:2393-2400]. An analysisof linked SEER-Medicare data between 2001 and 2005 found that 62% ofMedicare beneficiaries with MDS received ESAs [Davidoff et al. (2013)Leuk Res 37:675-680]. Greenberg et al. (2009) Blood 114:2393-2400]. Somepreclinical and clinical studies suggested that granulocytecolony-stimulating factor (G-CSF) can have synergistic effects withESAs, and small doses of G-CSF can be tried to improve erythroidresponses in some patients, especially those with RARS, either initiallyor in case of lack of response to sole ESA therapy [Negrin et al. (1996)Blood 87:4076-4081]. Additionally, patients with low-risk MDS and lowerlevels of serum EPO 200-500 mU/mL) and those who have lower RBCtransfusion requirements (<2 units/month) have higher probabilities ofachieving erythroid responses with ESAs [Hellstrom-Lindberg et al.(2003) Br J Haematol 120:1037-1046; Park et al. (2008) 111:574-582].Therefore, one or more ActRII antagonist agents of the disclosure (e.g.,a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide,a GDF trap, etc.) may be used in combination with granulocytecolony-stimulating factor (e.g., filgrastim) or granulocyte macrophagecolony-stimulating factor (e.g., sargramostim) to treat anemia.

In certain embodiments, the present disclosure provides methods oftreating or preventing anemia in an individual in need thereof byadministering to the individual a therapeutically effective amount ofone or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.) and a EPO receptor activator. In certain embodiments,one or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.) may be used in combination with EPO receptor activatorsto reduce the required dose of these activators in patients that aresusceptible to adverse effects of ESAs. These methods may be used fortherapeutic and prophylactic treatments of a patient.

One or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.) may be used in combination with EPO receptor activatorsto achieve an increase in red blood cells, particularly at lower doseranges. This may be beneficial in reducing the known off-target effectsand risks associated with high doses of EPO receptor activators. Theprimary adverse effects of ESAs include, for example, an excessiveincrease in the hematocrit or hemoglobin levels and polycythemia.Elevated hematocrit levels can lead to hypertension (more particularlyaggravation of hypertension) and vascular thrombosis. Other adverseeffects of ESAs which have been reported, some of which relate tohypertension, are headaches, influenza-like syndrome, obstruction ofshunts, myocardial infarctions and cerebral convulsions due tothrombosis, hypertensive encephalopathy, and red cell blood cellaplasia. See, e.g., Singibarti (1994) J. Clin Investig 72(suppl 6),S36-S43; Horl et al. (2000) Nephrol Dial Transplant 15(suppl 4), 51-56;Delanty et al. (1997) Neurology 49, 686-689; and Bunn (2002) N Engl JMed 346(7), 522-523).

Provided that antagonists of the present disclosure act by a differentmechanism that ESAs, these antagonists may be useful for increasing redblood cell and hemoglobin levels in patients that do not respond well toESAs or other EPO receptor activators. For example, an ActRII antagonistof the present disclosure may be beneficial for a patient in whichadministration of a normal to increased (>300 IU/kg/week) dose of ESAdoes not result in the increase of hemoglobin level up to the targetlevel. Patients with an inadequate response to ESAs are found in alltypes of anemia, but higher numbers of non-responders have been observedparticularly frequently in patients with cancers and patients withend-stage renal disease. An inadequate response to ESAs can be eitherconstitutive (observed upon the first treatment with ESA) or acquired(observed upon repeated treatment with ESA).

Lenalidomide is a thalidomide derivative approved for patients withlower-risk MDS with del5q and transfusion-dependent anemia. Pomalidomideis another thalidomide derivative. Approval of lenalidomide in the U.S.was based on results of a phase 2 trial in which transfusionindependence was achieved in about two thirds of patients studied, andthe mean duration of transfusion independence was 2.2 years [List et al.(2006) N Engl J Med 355:1456-1465]. Subsequently approved also in Europe[Giagounidis et al. (2014) Eur J Haematol 93:429-438], lenalidomide isconsidered the first-line treatment for patients with lower-risk MDSwith del5q and anemia. In certain embodiments, one or more ActRIIantagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, anActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc.),optionally combined with an EPO receptor activator and/or one or moreadditional therapies, may be used in combination with lenalidomide fortreating patients with MDS.

Aberrant DNA methylation is a poor prognostic feature in MDS [Shen e al.(2010) J Clin Oncol 28:605-613]. Azacitidine and decitabine are twoagents with DNA hypomethylating activity currently used to treatpatients mainly with high-risk MDS [Kantarjian et al. (2007) Cancer109:1133-1137; Fenaux et al. (2009) Lancet Oncol 10:223-232]. Since themechanism underlying their therapeutic effects is uncertain, theseagents are sometimes classified according to their chemical structure(azanucleosides) or known activity in vitro (DNA methyltransferaseinhibitors). Although there is less experience with azacitidine anddecitabine therapeutically in lower-risk MDS, studies indicate thatazacytidine and decitabine can produce an erythroid response in 30% to40% of ESA-resistant patients with lower-risk MDS [Lyons et al. (2009) JClin Oncol 27:1850-1856]. Platelet responses are also observed inthrombocytopenic patients. On the basis of these results, azacitidineand decitabine are approved in the United States for the treatment oflower-risk MDS with symptomatic cytopenias. In certain embodiments, oneor more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRIIantagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap,etc.), optionally combined with an EPO receptor activator and/or one ormore additional therapies, may be used in combination with azacitidine,decitabine, or another DNA methyltransferase inhibitor for treatingpatients with MDS.

Thrombocytopenia occurs in approximately 35% to 45% of MDS patients, whomay complain of easy bruising or frequent minor mucocutaneous bleedingand may display purpura or petechiae [Steensma et al. (2006) Mayo ClinProc 81:104-130]. In more extreme cases, increased risk ofgastrointestinal bleeding or intracranial hemorrhage may occur. Forthrombocytopenic patients, platelet transfusions are typically indicatedwhen platelet levels drop to less than 10,000 platelets/μL [Slichter(2007) Hematology Am Soc Hematol Educ Program 2007:172-178]. Thesupportive use of platelet transfusions is transient and not alwayseffective due to the frequency of sensitization in chronicallytransfusion-dependent MDS patients. There is considerable interest inusing growth factors with thrombopoietic activity in the therapy of MDS.Romiplostim and eltrombopag are thrombomimetic agents approved in theUnited States for patients with idiopathic thrombocytopenic purpura, andromiplostim is being studied extensively in patients with MDS[Kantarjian et al. (2010) J Clin Oncol 28:437-444; Santini (2012) SeminHematol 49:295-303]. When used as a single agent, romiplostim cansignificantly improve platelet counts in approximately 50% of patientswith lower-risk MDS with thrombocytopenia. However, a transient increasein marrow blast percentage, sometimes to greater than 20%, can beobserved in 15% of patients, consistent with the presence ofthrombopoietin receptors on blast cells in MDS. Romiplostim can alsosignificantly reduce thrombocytopenia and/or platelet transfusions inpatients with MDS receiving azacitidine, decitabine, or lenalidomide,thalidomide or pomalidomide, and could become an important adjunct tothose treatments [Kantarjian et al. (2010) Blood 116:3163-3170].Eltrombopag is also being developed in MDS. In certain embodiments, oneor more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRIIantagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap,etc.), optionally combined with an EPO receptor activator and/or one ormore additional therapies, may be used in combination withthrombomimetic agents such as romiplostim or eltrombopag for treatingpatients with MDS or sideroblastic anemias.

In certain embodiments, one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.), optionally combined with an EPOreceptor activator and/or one or more additional therapies, may be usedin combination with hepcidin, a hepcidin analog, or a hepcidin receptoractivator for treating patients with MDS or sideroblastic anemias,particularly for complications associated with iron overload. Acirculating polypeptide produced mainly in the liver, hepcidin isconsidered a master regulator of iron metabolism by virtue of itsability to induce the degradation of ferroportin, an iron-export proteinlocalized on absorptive enterocytes, hepatocytes, and macrophages. Inbroad terms, hepcidin reduces availability of extracellular iron, sohepcidin, hepcidin analogs, or hepcidin receptor activators may bebeneficial in the treatment of patients with MDS or sideroblasticanemias, particularly for complications associated with iron overload.

Investigational agents for MDS are in development. These includesingle-agent inhibitors of histone deacetylase, p38MAPK inhibitors,glutathione S-transferase 7C inhibitors, and alemtuzumab for patientswho meet criteria for immunosuppressive-based therapy [Garcia-Manero etal. (2011) J Clin Oncol 29:516-523;]. For example, high response rateshave been reported in MDS patients treated with immunosuppressivetherapies incorporating antithymocyte globulin or alemtuzumab [Sloand etal. (2010) J Clin Oncol 28:5166-5173]. However, such patients havegenerally been younger and have had a higher frequency of normalkaryotypes than MDS patients overall, which limits the generalizabilityof those results. Investigational therapies include, for example,histone deacetylase inhibitors (HDAC inhibitors), including vorinostat,valproic acid, phenylbutyrate, entinostat, MGCD0103, and other class Inuclear HDAC inhibitors, class II non-nuclear HDAC inhibitors, pan HDACinhibitors, and isoform-specific HDAC inhibitors; farnesyltransferaseinhibitors, including as tipifarnib and lonafarnib; tumor necrosisfactor-alpha (TNF-α) inhibitors, including etanercept or infliximab;inhibitors of glutathione-S-transferase (GST) P1-1, includingezatiostat; and inhibitors of CD33, including gemtuzumab ozogamicin. Incertain embodiments, one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.), optionally combined with an EPOreceptor activator and/or one or more additional therapies, may be usedin combination with one or more of these investigational therapies forMDS.

Increasing evidence suggests that combined use of MDS therapeutic agentswith different mechanisms of action offers substantial benefit in theform of diminished side effects, improved overall survival, and delayedprogression to acute myeloid leukemia. Multiple studies indicate thatwhen compared with traditional monotherapies, combining variousmedications with non-overlapping mechanisms of action and toxicities mayresult in significant benefit for MDS patients. A variety of combinationtherapies with growth factors, DNA methytransferase inhibitors, histonedeacetylase inhibitors, and immunosuppressant treatments provideencouraging data [Ornstein et al. (2012) Int J Hematol 95:26-33].Therefore, one or more ActRII antagonist agents of the disclosure (e.g.,a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide,a GDF trap, etc.), optionally combined with an EPO receptor activator,may be used to increase numbers of red blood cells in MDS patientsadditionally treated with a combination of two or more agents, includingbut not limited to growth factors, DNA methyltransferase inhibitors,histone deacetylase inhibitors, or immunosuppressant treatments.

In certain embodiments, the present disclosure provides methods formanaging a patient that has been treated with, or is a candidate to betreated with, one or more one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.) by measuring one or morehematologic parameters in the patient. The hematologic parameters may beused to evaluate appropriate dosing for a patient who is a candidate tobe treated with the antagonist of the present disclosure, to monitor thehematologic parameters during treatment, to evaluate whether to adjustthe dosage during treatment with one or more antagonist of thedisclosure, and/or to evaluate an appropriate maintenance dose of one ormore antagonists of the disclosure. If one or more of the hematologicparameters are outside the normal level, dosing with one or more ActRIIantagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, anActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc.) may bereduced, delayed or terminated.

Hematologic parameters that may be measured in accordance with themethods provided herein include, for example, red blood cell levels,blood pressure, iron stores, and other agents found in bodily fluidsthat correlate with increased red blood cell levels, using artrecognized methods. Such parameters may be determined using a bloodsample from a patient. Increases in red blood cell levels, hemoglobinlevels, and/or hematocrit levels may cause increases in blood pressure.

In one embodiment, if one or more hematologic parameters are outside thenormal range or on the high side of normal in a patient who is acandidate to be treated with one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.), then onset of administration ofthe one or more antagonists of the disclosure may be delayed until thehematologic parameters have returned to a normal or acceptable leveleither naturally or via therapeutic intervention. For example, if acandidate patient is hypertensive or pre-hypertensive, then the patientmay be treated with a blood pressure lowering agent in order to reducethe patient's blood pressure. Any blood pressure lowering agentappropriate for the individual patient's condition may be usedincluding, for example, diuretics, adrenergic inhibitors (includingalpha blockers and beta blockers), vasodilators, calcium channelblockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensinII receptor blockers. Blood pressure may alternatively be treated usinga diet and exercise regimen. Similarly, if a candidate patient has ironstores that are lower than normal, or on the low side of normal, thenthe patient may be treated with an appropriate regimen of diet and/oriron supplements until the patient's iron stores have returned to anormal or acceptable level. For patients having higher than normal redblood cell levels and/or hemoglobin levels, then administration of theone or more antagonists of the disclosure may be delayed until thelevels have returned to a normal or acceptable level.

In certain embodiments, if one or more hematologic parameters areoutside the normal range or on the high side of normal in a patient whois a candidate to be treated with one or more ActRII antagonist agentsof the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIApolypeptide, an ActRIIB polypeptide, a GDF trap, etc.), then the onsetof administration may not be delayed. However, the dosage amount orfrequency of dosing of the one or more antagonists of the disclosure maybe set at an amount that would reduce the risk of an unacceptableincrease in the hematologic parameters arising upon administration ofthe one or more antagonists of the disclosure. Alternatively, atherapeutic regimen may be developed for the patient that combines oneor more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRIIantagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap,etc.) with a therapeutic agent that addresses the undesirable level ofthe hematologic parameter. For example, if the patient has elevatedblood pressure, then a therapeutic regimen may be designed involvingadministration of one or more ActRII antagonist agents of the disclosure(e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIBpolypeptide, a GDF trap, etc.) and a blood pressure lowering agent. Fora patient having lower than desired iron stores, a therapeutic regimenmay be developed involving one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.) and iron supplementation.

In one embodiment, baseline parameter(s) for one or more hematologicparameters may be established for a patient who is a candidate to betreated with one or more ActRII antagonist agents of the disclosure(e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIBpolypeptide, a GDF trap, etc.) and an appropriate dosing regimenestablished for that patient based on the baseline value(s).Alternatively, established baseline parameters based on a patient'smedical history could be used to inform an appropriate antagonist dosingregimen for a patient. For example, if a healthy patient has anestablished baseline blood pressure reading that is above the definednormal range it may not be necessary to bring the patient's bloodpressure into the range that is considered normal for the generalpopulation prior to treatment with the one or more antagonist of thedisclosure. A patient's baseline values for one or more hematologicparameters prior to treatment with one or more ActRII antagonist agentsof the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIApolypeptide, an ActRIIB polypeptide, a GDF trap, etc.) may also be usedas the relevant comparative values for monitoring any changes to thehematologic parameters during treatment with the one or more antagonistsof the disclosure.

In certain embodiments, one or more hematologic parameters are measuredin patients who are being treated with a one or more ActRII antagonistagents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIApolypeptide, an ActRIIB polypeptide, a GDF trap, etc.). The hematologicparameters may be used to monitor the patient during treatment andpermit adjustment or termination of the dosing with the one or moreantagonists of the disclosure or additional dosing with anothertherapeutic agent. For example, if administration of one or more ActRIIantagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, anActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc.) resultsin an increase in blood pressure, red blood cell level, or hemoglobinlevel, or a reduction in iron stores, then the dose of the one or moreantagonists of the disclosure may be reduced in amount or frequency inorder to decrease the effects of the one or more antagonists of thedisclosure on the one or more hematologic parameters. If administrationof one or more ActRII antagonist agents of the disclosure (e.g., aGDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, aGDF trap, etc.) results in a change in one or more hematologicparameters that is adverse to the patient, then the dosing of the one ormore antagonists of the disclosure may be terminated either temporarily,until the hematologic parameter(s) return to an acceptable level, orpermanently. Similarly, if one or more hematologic parameters are notbrought within an acceptable range after reducing the dose or frequencyof administration of the one or more antagonists of the disclosure, thenthe dosing may be terminated. As an alternative, or in addition to,reducing or terminating the dosing with the one or more antagonists ofthe disclosure, the patient may be dosed with an additional therapeuticagent that addresses the undesirable level in the hematologicparameter(s), such as, for example, a blood pressure lowering agent oran iron supplement. For example, if a patient being treated with one ormore ActRII antagonist agents of the disclosure (e.g., a GDF-ActRIIantagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap,etc.) has elevated blood pressure, then dosing with the one or moreantagonists of the disclosure may continue at the same level and ablood-pressure-lowering agent is added to the treatment regimen, dosingwith the one or more antagonist of the disclosure may be reduced (e.g.,in amount and/or frequency) and a blood-pressure-lowering agent is addedto the treatment regimen, or dosing with the one or more antagonist ofthe disclosure may be terminated and the patient may be treated with ablood-pressure-lowering agent.

6. Pharmaceutical Compositions

In certain aspects, one or more ActRII antagonist agents of thedisclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, anActRIIB polypeptide, a GDF trap, etc.) can be administered alone or as acomponent of a pharmaceutical formulation (also referred to as atherapeutic composition or pharmaceutical composition). A pharmaceuticalformulation refers to a preparation which is in such form as to permitthe biological activity of an active ingredient (e.g., an agent of thepresent disclosure) contained therein to be effective and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. The subject compounds maybe formulated for administration in any convenient way for use in humanor veterinary medicine. For example, one or more agents of the presentdisclosure may be formulated with a pharmaceutically acceptable carrier.A pharmaceutically acceptable carrier refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isgenerally nontoxic to a subject. A pharmaceutically acceptable carrierincludes, but is not limited to, a buffer, excipient, stabilizer, and/orpreservative. In general, pharmaceutical formulations for use in thepresent disclosure are in a pyrogen-free, physiologically-acceptableform when administered to a subject. Therapeutically useful agents otherthan those described herein, which may optionally be included in theformulation as described above, may be administered in combination withthe subject agents in the methods of the present disclosure.

Typically, compounds will be administered parenterally [e.g., byintravenous (I.V.) injection, intraarterial injection, intraosseousinjection, intramuscular injection, intrathecal injection, subcutaneousinjection, or intradermal injection]. Pharmaceutical compositionssuitable for parenteral administration may comprise one or more agentsof the disclosure in combination with one or more pharmaceuticallyacceptable sterile isotonic aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, or sterile powders which may bereconstituted into sterile injectable solutions or dispersions justprior to use. Injectable solutions or dispersions may containantioxidants, buffers, bacteriostats, suspending agents, thickeningagents, or solutes which render the formulation isotonic with the bloodof the intended recipient. Examples of suitable aqueous and nonaqueouscarriers which may be employed in the pharmaceutical formulations of thepresent disclosure include water, ethanol, polyols (e.g., glycerol,propylene glycol, polyethylene glycol, etc.), vegetable oils (e.g.,olive oil), injectable organic esters (e.g., ethyl oleate), and suitablemixtures thereof. Proper fluidity can be maintained, for example, by theuse of coating materials (e.g., lecithin), by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In some embodiments, a therapeutic method of the present disclosureincludes administering the pharmaceutical composition systemically, orlocally, from an implant or device. Further, the pharmaceuticalcomposition may be encapsulated or injected in a form for delivery to atarget tissue site (e.g., bone marrow or muscle). In certainembodiments, compositions of the present disclosure may include a matrixcapable of delivering one or more of the agents of the presentdisclosure to a target tissue site (e.g., bone marrow or muscle),providing a structure for the developing tissue and optimally capable ofbeing resorbed into the body. For example, the matrix may provide slowrelease of one or more agents of the present disclosure. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material may be based on one or more of:biocompatibility, biodegradability, mechanical properties, cosmeticappearance, and interface properties. The particular application of thesubject compositions will define the appropriate formulation. Potentialmatrices for the compositions may be biodegradable and chemicallydefined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylacticacid, and polyanhydrides. Other potential materials are biodegradableand biologically well-defined, including, for example, bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenon-biodegradable and chemically defined, including, for example,sintered hydroxyapatite, bioglass, aluminates, or other ceramics.Matrices may be comprised of combinations of any of the above mentionedtypes of material including, for example, polylactic acid andhydroxyapatite or collagen and tricalciumphosphate. The bioceramics maybe altered in composition (e.g., calcium-aluminate-phosphate) andprocessing to alter one or more of pore size, particle size, particleshape, and biodegradability.

In certain embodiments, pharmaceutical compositions of the presentdisclosure can be administered orally, for example, in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis suchas sucrose and acacia or tragacanth), powders, granules, a solution or asuspension in an aqueous or non-aqueous liquid, an oil-in-water orwater-in-oil liquid emulsion, or an elixir or syrup, or pastille (usingan inert base, such as gelatin and glycerin, or sucrose and acacia),and/or a mouth wash, each containing a predetermined amount of acompound of the present disclosure and optionally one or more otheractive ingredients. A compound of the present disclosure and optionallyone or more other active ingredients may also be administered as abolus, electuary, or paste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, and granules), one or more compounds of thepresent disclosure may be mixed with one or more pharmaceuticallyacceptable carriers including, for example, sodium citrate, dicalciumphosphate, a filler or extender (e.g., a starch, lactose, sucrose,glucose, mannitol, and silicic acid), a binder (e.g.carboxymethylcellulose, an alginate, gelatin, polyvinyl pyrrolidone,sucrose, and acacia), a humectant (e.g., glycerol), a disintegratingagent (e.g., agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, a silicate, and sodium carbonate), a solution retardingagent (e.g. paraffin), an absorption accelerator (e.g. a quaternaryammonium compound), a wetting agent (e.g., cetyl alcohol and glycerolmonostearate), an absorbent (e.g., kaolin and bentonite clay), alubricant (e.g., a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), a coloring agent, andmixtures thereof. In the case of capsules, tablets, and pills, thepharmaceutical formulation (composition) may also comprise a bufferingagent. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using one or moreexcipients including, e.g., lactose or a milk sugar as well as a highmolecular-weight polyethylene glycol.

Liquid dosage forms for oral administration of the pharmaceuticalcomposition may include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredient(s), the liquid dosage form may contain an inertdiluent commonly used in the art including, for example, water or othersolvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil (e.g.,cottonseed, groundnut, corn, germ, olive, castor, and sesame oil),glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acidester of sorbitan, and mixtures thereof]. Besides inert diluents, theoral formulation can also include an adjuvant including, for example, awetting agent, an emulsifying and suspending agent, a sweetening agent,a flavoring agent, a coloring agent, a perfuming agent, a preservativeagent, and combinations thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents including, for example, an ethoxylated isostearyl alcohol,polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar, tragacanth, andcombinations thereof.

Prevention of the action and/or growth of microorganisms may be ensuredby the inclusion of various antibacterial and antifungal agentsincluding, for example, paraben, chlorobutanol, and phenol sorbic acid.

In certain embodiments, it may be desirable to include an isotonic agentincluding, for example, a sugar or sodium chloride into thecompositions. In addition, prolonged absorption of an injectablepharmaceutical form may be brought about by the inclusion of an agentthat delays absorption, including, for example, aluminum monostearateand gelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the one or more of the agents of the present disclosure. The variousfactors include, but are not limited to, the patient's red blood cellcount, hemoglobin level, the desired target red blood cell count, thepatient's age, the patient's sex, the patient's diet, the severity ofany disease that may be contributing to a depressed red blood celllevel, the time of administration, and other clinical factors. Theaddition of other known active agents to the final composition may alsoaffect the dosage. Progress can be monitored by periodic assessment ofone or more of red blood cell levels, hemoglobin levels, reticulocytelevels, and other indicators of the hematopoietic process.

In certain embodiments, the present disclosure also provides genetherapy for the in vivo production of one or more of the agents of thepresent disclosure. Such therapy would achieve its therapeutic effect byintroduction of the agent sequences into cells or tissues having one ormore of the disorders as listed above. Delivery of the agent sequencescan be achieved, for example, by using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Preferredtherapeutic delivery of one or more of agent sequences of the disclosureis the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus(e.g., a retrovirus). The retroviral vector may be a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous sarcoma virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. Retroviral vectors can be made target-specific by attaching,for example, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing one or more of theagents of the present disclosure.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes (gag, pol, and env),by conventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for one or more of the agents of thepresent disclosure is a colloidal dispersion system. Colloidaldispersion systems include, for example, macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Incertain embodiments, the preferred colloidal system of this disclosureis a liposome. Liposomes are artificial membrane vesicles which areuseful as delivery vehicles in vitro and in vivo. RNA, DNA, and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form [see, e.g., Fraley, et al. (1981)Trends Biochem. Sci., 6:77]. Methods for efficient gene transfer using aliposome vehicle are known in the art [see, e.g., Mannino, et al. (1988)Biotechniques, 6:682, 1988].

The composition of the liposome is usually a combination ofphospholipids, which may include a steroid (e.g. cholesterol). Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Other phospholipids or other lipidsmay also be used, including, for example a phosphatidyl compound (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, sphingolipid, cerebroside, or a ganglioside),egg phosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ specificity, cell specificity, andorganelle specificity and is known in the art.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1: ActRIIa-Fc Fusion Proteins

Applicants constructed a soluble ActRIIA fusion protein that has theextracellular domain of human ActRIIa fused to a human or mouse Fcdomain with a minimal linker in between. The constructs are referred toas ActRIIA-hFc and ActRIIA-mFc, respectively.

ActRIIA-hFc is shown below as purified from CHO cell lines (SEQ IDNO:22):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIA-hFc and ActRIIA-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 23) MKFLVNVALVFMVVYISYIYA(ii) Tissue plasminogen activator (TPA): (SEQ ID NO: 24)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 25)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 26) MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence:

(SEQ ID NO: 27) ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGAATTC

Both ActRIIA-hFc and ActRIIA-mFc were remarkably amenable to recombinantexpression. As shown in FIG. 3, the protein was purified as a single,well-defined peak of protein. N-terminal sequencing revealed a singlesequence of -ILGRSETQE (SEQ ID NO:34). Purification could be achieved bya series of column chromatography steps, including, for example, threeor more of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. TheActRIIA-hFc protein was purified to a purity of >98% as determined bysize exclusion chromatography and >95% as determined by SDS PAGE.

ActRIIA-hFc and ActRIIA-mFc showed a high affinity for ligands,particularly activin A. GDF-11 or activin A were immobilized on aBiacore™ CM5 chip using standard amine-coupling procedure. ActRIIA-hFcand ActRIIA-mFc proteins were loaded onto the system, and binding wasmeasured. ActRIIA-hFc bound to activin with a dissociation constant(K_(D)) of 5×10⁻¹² and bound to GDF11 with a K_(D) of 9.96×10⁻⁹. SeeFIG. 4. ActRIIA-mFc behaved similarly.

The ActRIIA-hFc was very stable in pharmacokinetic studies. Rats weredosed with 1 mg/kg, 3 mg/kg, or 10 mg/kg of ActRIIA-hFc protein, andplasma levels of the protein were measured at 24, 48, 72, 144 and 168hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg, or 30mg/kg. In rats, ActRIIA-hFc had an 11-14 day serum half-life, andcirculating levels of the drug were quite high after two weeks (11μg/ml, 110 μg/ml, or 304 μg/ml for initial administrations of 1 mg/kg,10 mg/kg, or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasmahalf-life was substantially greater than 14 days, and circulating levelsof the drug were 25 μg/ml, 304 μg/ml, or 1440 μg/ml for initialadministrations of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively.

Example 2: Characterization of an ActRIIA-hFc Protein

ActRIIA-hFc fusion protein was expressed in stably transfected CHO-DUKXB11 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using atissue plasminogen leader sequence of SEQ ID NO:9. The protein, purifiedas described above in Example 1, had a sequence of SEQ ID NO:22. The Fcportion is a human IgG1 Fc sequence, as shown in SEQ ID NO:22. Proteinanalysis reveals that the ActRIIA-hFc fusion protein is formed as ahomodimer with disulfide bonding.

The CHO-cell-expressed material has a higher affinity for activin Bligand than that reported for an ActRIIa-hFc fusion protein expressed inhuman 293 cells [see, del Re et al. (2004) J Biol Chem.279(51):53126-53135]. Additionally, the use of the TPA leader sequenceprovided greater production than other leader sequences and, unlikeActRIIA-Fc expressed with a native leader, provided a highly pureN-terminal sequence. Use of the native leader sequence resulted in twomajor species of ActRIIA-Fc, each having a different N-terminalsequence.

Example 3. ActRIIA-hFc Increases Red Blood Cell Levels in Non-HumanPrimates

The study employed four groups of five male and five female cynomolgusmonkeys each, with three per sex per group scheduled for termination onDay 29, and two per sex per group scheduled for termination on Day 57.Each animal was administered the vehicle (Group 1) or ActRIIA-Fc atdoses of 1, 10, or 30 mg/kg (Groups 2, 3 and 4, respectively) viaintravenous (IV) injection on Days 1, 8, 15, and 22. The dose volume wasmaintained at 3 mL/kg. Various measures of red blood cell levels wereassessed two days prior to the first administration and at days 15, 29,and 57 (for the remaining two animals) after the first administration.

The ActRIIA-hFc caused statistically significant increases in mean redblood cell parameters [red blood cell count (RBC), hemoglobin (HGB), andhematocrit (HCT)] for males and females, at all dose levels and timepoints throughout the study, with accompanying elevations in absoluteand relative reticulocyte counts (ARTC; RTC). See FIGS. 5-8.

Statistical significance was calculated for each treatment grouprelative to the mean for the treatment group at baseline.

Notably, the increases in red blood cell counts and hemoglobin levelsare roughly equivalent in magnitude to effects reported witherythropoietin. The onset of these effects is more rapid with ActRIIA-Fcthan with erythropoietin.

Similar results were observed with rats and mice.

Example 4: ActRIIA-hFc Increases Red Blood Cell Levels and Markers ofBone Formation in Human Patients

The ActRIIA-hFc fusion protein described in Example 1 was administeredto human subjects in a randomized, double-blind, placebo-controlledstudy that was conducted to evaluate, primarily, the safety of theprotein in healthy, postmenopausal women. Forty-eight subjects wererandomized in cohorts of 6 to receive either a single dose ofActRIIA-hFc or placebo (5 active:1 placebo). Dose levels ranged from0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.1 mg/kgsubcutaneously (SC). All subjects were followed for 120 days. Inaddition to pharmacokinetic (PK) analyses, the biologic activity ofActRIIA-hFc was also assessed by measurement of biochemical markers ofbone formation and resorption as well as FSH levels.

To look for potential changes, hemoglobin and RBC numbers were examinedin detail for all subjects over the course of the study and compared tothe baseline levels. Platelet counts were compared over the same time asthe control. There were no clinically significant changes from thebaseline values over time for the platelet counts.

Pharmacokinetic (PK) analysis of ActRIIA-hFc revealed a linear profilewith dose, and a mean half-life of approximately 25-32 days. Thearea-under-curve (AUC) for ActRIIA-hFc was linearly related to dose, andthe absorption after SC dosing was essentially complete. See FIGS. 9 and10. These data indicate that SC is a desirable approach to dosingbecause it provides equivalent bioavailability and serum-half life forthe drug while avoiding the spike in serum concentrations of drugassociated with the first few days of IV dosing (see FIG. 10).ActRIIA-hFc caused a rapid, sustained dose-dependent increase in serumlevels of bone-specific alkaline phosphatase (BAP), which is a markerfor anabolic bone growth, and a dose-dependent decrease in C-terminaltype 1 collagen telopeptide and tartrate-resistant acid phosphatase 5blevels, which are markers for bone resorption. Other markers such asP1NP showed inconclusive results. BAP levels showed near-saturatingeffects at the highest dosage of drug, indicating that half-maximaleffects on this anabolic bone biomarker could be achieved at a dosage of0.3 mg/kg, with increases ranging up to 3 mg/kg. Calculated as arelationship of pharmacodynamic effect to AUC for drug, the EC50 was51,465 (day*ng/ml) (see FIG. 11). These bone biomarker changes weresustained for approximately 120 days at the highest dose levels tested.There was also a dose-dependent decrease in serum FSH levels consistentwith inhibition of activin.

Overall, there was a very small non-drug related reduction in hemoglobinover the first week of the study probably related to study phlebotomy inthe 0.01 and 0.03 mg/kg groups whether given IV or SC. The 0.1 mg/kg SCand IV hemoglobin results were stable or showed modest increases by Day8-15. At the 0.3 mg/kg IV dose level there was a clear increase in HGBlevels seen as early as Day 2 and often peaking at Day 15-29 that wasnot seen in the placebo-treated subjects. At the 1.0 mg/kg IV dose andthe 3.0 mg/kg IV dose, mean increases in hemoglobin of greater than 1g/dl were observed in response to the single dose, with correspondingincreases in RBC counts and hematocrit. These hematologic parameterspeaked at about 60 days after the dose and substantial decrease by day120. This indicates that dosing for the purpose of increasing red bloodcell levels may be more effective if done at intervals less than 120days (i.e., prior to return to baseline), with dosing intervals of 90days or less or 60 days or less may be desirable. For a summary ofhematological changes, see FIGS. 12-15.

Overall, ActRIIA-hFc showed a dose-dependent effect on red blood cellcounts and reticulocyte counts.

Example 5: Treatment of an Anemic Patient with ActRIIA-hFc

A clinical study was designed to treat patients with multiple doses ofActRIIA-hFc, at three dose levels of 0.1 mg/kg, 0.3 mg/kg, and 1.0mg/kg, with dosing to occur every 30 days. Normal healthy subjects inthe trial exhibited an increase in hemoglobin and hematocrit that isconsistent with the increases seen in the Phase I clinical trialreported in Example 4, except that in some instances the hemoglobin (Hg)and hematocrit (Hct) are elevated beyond the normal range. An anemicpatient with hemoglobin levels of approximately 7.5 g/dL also receivedtwo doses at the 1 mg/kg level, resulting in a hemoglobin level ofapproximately 10.5 g/dL after two months. The patient's anemia was amicrocytic anemia, thought to be caused by chronic iron deficiency.

ActRIIA-Fc fusion proteins have been further demonstrated to beeffective in increasing red blood cell levels in various models ofanemia including, for example, chemotherapy-induced anemia and anemiaassociated with chronic kidney disease (see, e.g., U.S. PatentApplication Publication No. 2010/0028331).

Example 6: Alternative ActRIIA-Fc Proteins

A variety of ActRIIA variants that may be used according to the methodsdescribed herein are described in the International Patent Applicationpublished as WO2006/012627 (see e.g., pp. 55-58), incorporated herein byreference in its entirety. An alternative construct may have a deletionof the C-terminal tail (the final 15 amino acids of the extracellulardomain of ActRIIA. The sequence for such a construct is presented below(Fc portion underlined) (SEQ ID NO:28):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 7: Generation of ActRIIB-Fc Fusion Proteins

Applicants constructed a soluble ActRIIB fusion protein that has theextracellular domain of human ActRIIB fused to a human or mouse Fcdomain with a minimal linker (three glycine amino acids) in between. Theconstructs are referred to as ActRIIB-hFc and ActRIIB-mFc, respectively.

ActRIIB-hFc is shown below as purified from CHO cell lines (SEQ IDNO:29):

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIB-hFc and ActRIIB-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 23) MKFLVNVALVFMVVYISYIYA(ii) Tissue plasminogen activator (TPA): (SEQ ID NO: 24)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 30)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence (SEQ ID NO: 31):

MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQID NO:32):

A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTTTCGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCAACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGACAAGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGAAGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGGAGAACCCCCA GGTGTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTCATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA GCCCCCACCGGTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAGTCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCACATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGGACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGTACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACAAGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAAGCCAAAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCAAGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGGAGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGGGGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGAGCCTCTCCCT GTCTCCGGGT AAATGA

N-terminal sequencing of the CHO-cell-produced material revealed a majorsequence of -GRGEAE (SEQ ID NO:33). Notably, other constructs reportedin the literature begin with an -SGR . . . sequence.

Purification could be achieved by a series of column chromatographysteps, including, for example, three or more of the following, in anyorder: protein A chromatography, Q sepharose chromatography,phenylsepharose chromatography, size exclusion chromatography, andcation exchange chromatography. The purification could be completed withviral filtration and buffer exchange.

ActRIIB-Fc fusion proteins were also expressed in HEK293 cells and COScells. Although material from all cell lines and reasonable cultureconditions provided protein with muscle-building activity in vivo,variability in potency was observed perhaps relating to cell lineselection and/or culture conditions.

Applicants generated a series of mutations in the extracellular domainof ActRIIB and produced these mutant proteins as soluble fusion proteinsbetween extracellular ActRIIB and an Fc domain. The backgroundActRIIB-Fc fusion has the sequence of SEQ ID NO:29.

Various mutations, including N- and C-terminal truncations, wereintroduced into the background ActRIIB-Fc protein. Based on the datapresented in Example 1, it is expected that these constructs, ifexpressed with a TPA leader, will lack the N-terminal serine. Mutationswere generated in ActRIIB extracellular domain by PCR mutagenesis. AfterPCR, fragments were purified through a Qiagen column, digested with SfoIand AgeI and gel purified. These fragments were ligated into expressionvector pAID4 (see WO2006/012627) such that upon ligation it createdfusion chimera with human IgG1. Upon transformation into E. coli DH5alpha, colonies were picked and DNAs were isolated. For murineconstructs (mFc), a murine IgG2a was substituted for the human IgG1.Sequences of all mutants were verified.

All of the mutants were produced in HEK293T cells by transienttransfection. In summary, in a 500 ml spinner, HEK293T cells were set upat 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 ml volume andgrown overnight. Next day, these cells were treated with DNA:PEI (1:1)complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml mediawas added and cells were grown for 7 days. Conditioned media washarvested by spinning down the cells and concentrated.

Mutants were purified using a variety of techniques, including, forexample, a protein A column, and eluted with low pH (3.0) glycinebuffer. After neutralization, these were dialyzed against PBS.

Mutants were also produced in CHO cells by similar methodology. Mutantswere tested in binding assays and/or bioassays described in WO2008/097541 and WO 2006/012627 incorporated by reference herein. In someinstances, assays were performed with conditioned medium rather thanpurified proteins. Additional variations of ActRIIB are described inU.S. Pat. No. 7,842,663.

Example 8: ActRIIB-Fc Stimulates Erythropoiesis in Non-Human Primates

Cynomolgus monkeys were allocated into seven groups (6/sex/group) andadministered ActRIIB(20-134)-hFc as a subcutaneous injection at dosagesof 0.6, 3, or 15 mg/kg every 2 weeks or every 4 weeks over a 9-monthperiod. The control group (6/sex/group) received the vehicle at the samedose volume (0.5 ml/kg/dose) as ActRIIB(20-134)-hFc-treated animals.Animals were monitored for changes in general clinical pathologyparameters (e.g., hematology, clinical chemistry, coagulation, andurinalysis). Hematology, coagulation, and clinical chemistry parameters(including iron parameters, lipase, and amylase) were evaluated twiceprior to initiation of dosing and on Days 59, 143, 199, 227, and on Days267 (for groups dosed every 4 weeks) or 281 (for groups dosed every 2weeks). The evaluations on Days 267/281 occurred 2 weeks after the finaldose was administered.

Administration of ActRIIB(20-134)-hFc resulted in non-adverse,dose-related changes to hematology parameters in male and femalemonkeys. These changes included increased red blood cell count,reticulocyte count and red cell distribution width and decreased meancorpuscular volume, mean corpuscular hemoglobin, and platelet count. Inmales, RBC count was increased at all dose levels, and the magnitude ofincrease was generally comparable whether ActRIIB(20-134)-hFc wasadministered every 2 weeks or every 4 weeks. Mean RBC count wasincreased at all time points between Days 59 and 267/281 (except RBCcount was not increased in group 2 males [0.6 mg/kg every 2 weeks] onDay 281). In females, RBC count was increased at ≥3 mg/kg every 2 weeksand the changes occurred between Days 143 and 281; at 15 mg/kg every 4weeks mean RBC count was increased between Days 59 and 267.

These effects are consistent with a positive effect ofActRIIB(20-134)-hFc on stimulating erythropoiesis.

Example 9: Generation of a GDF Trap

Applicants constructed a GDF trap as follows. A polypeptide having amodified extracellular domain of ActRIIB (amino acids 20-134 of SEQ IDNO:1 with an L79D substitution) with greatly reduced activin A bindingrelative to GDF11 and/or myostatin (as a consequence of aleucine-to-aspartate substitution at position 79 in SEQ ID NO:1) wasfused to a human or mouse Fc domain with a minimal linker (three glycineamino acids) in between. The constructs are referred to as ActRIIB(L79D20-134)-hFc and ActRIIB(L79D 20-134)-mFc, respectively. Alternativeforms with a glutamate rather than an aspartate at position 79 performedsimilarly (L79E). Alternative forms with an alanine rather than a valineat position 226 with respect to SEQ ID NO:36, below were also generatedand performed equivalently in all respects tested. The aspartate atposition 79 (relative to SEQ ID NO: 1, or position 60 relative to SEQ IDNO:36) is indicated with double underlining below. The valine atposition 226 relative to SEQ ID NO:36 is also indicated by doubleunderlining below.

The GDF trap ActRIIB(L79D 20-134)-hFc is shown below as purified fromCHO cell lines (SEQ ID NO:36).

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP V PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIB-derived portion of the GDF trap has an amino acid sequenceset forth below (SEQ ID NO: 37), and that portion could be used as amonomer or as a non-Fc fusion protein as a monomer, dimer, orgreater-order complex.

(SEQ ID NO: 37) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT

The GDF trap protein was expressed in CHO cell lines. Three differentleader sequences were considered:

(i) Honey bee melittin (HBML): (SEQ ID NO: 23) MKFLVNVALVFMVVYISYIYA(ii) Tissue plasminogen activator (TPA): (SEQ ID NO: 24)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 30)MTAPWVALALLWGSLCAGS.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 38) MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQID NO:39):

A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTTTCGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCAACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGACAAGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGAAGGGCTGCTG GGACGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGGAGAACCCCCA GGTGTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTCATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA GCCCCCACCGGTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAGTCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCACATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGGACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGTACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACAAGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAAGCCAAAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCAAGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGGAGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGGGGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGAGCCTCTCCCT GTCTCCGGGT AAATGA

Purification could be achieved by a series of column chromatographysteps, including, for example, three or more of the following, in anyorder: protein A chromatography, Q sepharose chromatography,phenylsepharose chromatography, size exclusion chromatography, andcation exchange chromatography. The purification could be completed withviral filtration and buffer exchange. In an example of a purificationscheme, the cell culture medium is passed over a protein A column,washed in 150 mM Tris/NaCl (pH 8.0), then washed in 50 mM Tris/NaCl (pH8.0) and eluted with 0.1 M glycine, pH 3.0. The low pH eluate is kept atroom temperature for 30 minutes as a viral clearance step. The eluate isthen neutralized and passed over a Q-sepharose ion-exchange column andwashed in 50 mM Tris pH 8.0, 50 mM NaCl, and eluted in 50 mM Tris pH8.0, with an NaCl concentration between 150 mM and 300 mM. The eluate isthen changed into 50 mM Tris pH 8.0, 1.1 M ammonium sulfate and passedover a phenyl sepharose column, washed, and eluted in 50 mM Tris pH 8.0with ammonium sulfate between 150 and 300 mM. The eluate is dialyzed andfiltered for use.

Additional GDF traps (ActRIIB-Fc fusion proteins modified so as toreduce the ratio of activin A binding relative to myostatin or GDF11binding) are described in WO 2008/097541 and WO 2006/012627,incorporated by reference herein.

Example 10: Bioassay for GDF-11- and Activin-Mediated Signaling

An A-204 reporter gene assay was used to evaluate the effects ofActRIIB-Fc proteins and GDF traps on signaling by GDF-11 and activin A.Cell line: human rhabdomyosarcoma (derived from muscle). Reportervector: pGL3(CAGA)12 (described in Dennler et al, 1998, EMBO 17:3091-3100). The CAGA12 motif is present in TGF-beta responsive genes(e.g., PAI-1 gene), so this vector is of general use for factorssignaling through SMAD2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 orpGL3(CAGA)12(10 ug)+pRLCMV (1 μg) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with factors for 1 hr before adding to cells. Six hrslater, cells were rinsed with PBS and lysed.

This is followed by a luciferase assay. In the absence of anyinhibitors, activin A showed 10-fold stimulation of reporter geneexpression and an ED50˜2 ng/ml. GDF-11: 16 fold stimulation, ED50: ˜1.5ng/ml.

ActRIIB(20-134) is a potent inhibitor of activin A, GDF-8, and GDF-11activity in this assay. As described below, ActRIIB variants were alsotested in this assay.

Example 11: ActRIIB-Fc Variants, Cell-Based Activity

Activity of ActRIIB-Fc proteins and GDF traps was tested in a cell-basedassay as described above. Results are summarized in the table below.Some variants were tested in different C-terminal truncation constructs.As discussed above, truncations of five or fifteen amino acids causedreduction in activity. The GDF traps (L79D and L79E variants) showedsubstantial loss of activin A inhibition while retaining almostwild-type inhibition of GDF-11.

Soluble ActRIIB-Fc Binding to GDF11 and Activin A:

Portion of ActRIIB ActRIIB-Fc (corresponds to amino GDF11 InhibitionActivin Inhibition Variations acids of SEQ ID NO: 1) Activity ActivityR64 20-134 +++ +++ (approx. 10⁻⁸ M K_(I)) (approx. 10⁻⁸ M K_(I)) A6420-134 + + (approx. 10⁻⁶ M K_(I)) (approx. 10⁻⁶ M K_(I)) R64 20-129 ++++++ R64 K74A 20-134 ++++ ++++ R64 A24N 20-134 +++ +++ R64 A24N 20-119 ++++ R64 A24N K74A 20-119 + + R64 L79P 20-134 + + R64 L79P K74A 20-134 + +R64 L79D 20-134 +++ + R64 L79E 20-134 +++ + R64K 20-134 +++ +++ R64K20-129 +++ +++ R64 P129S P130A 20-134 +++ +++ R64N 20-134 + + + Pooractivity (roughly 1 × 10⁻⁶ K_(I)) ++ Moderate activity (roughly 1 × 10⁻⁷K_(I)) +++ Good (wild-type) activity (roughly 1 × 10⁻⁸ K_(I)) ++++Greater than wild-type activity

Several variants have been assessed for serum half-life in rats.ActRIIB(20-134)-Fc has a serum half-life of approximately 70 hours.ActRIIB(A24N 20-134)-Fc has a serum half-life of approximately 100-150hours. The A24N variant has activity in the cell-based assay (above) andin vivo assays (below) that is equivalent to the wild-type molecule.Coupled with the longer half-life, this means that over time an A24Nvariant will give greater effect per unit of protein than the wild-typemolecule. The A24N variant, and any of the other variants tested above,may be combined with the GDF trap molecules, such as the L79D or L79Evariants.

Example 12: GDF-11 and Activin a Binding

Binding of certain ActRIIB-Fc proteins and GDF traps to ligands wastested in a Biacore™ assay.

The ActRIIB-Fc variants or wild-type protein were captured onto thesystem using an anti-hFc antibody. Ligands were injected and flowed overthe captured receptor proteins. Results are summarized in the tablesbelow.

Ligand-binding specificity IIB variants. Protein Kon (1/Ms) Koff (1/s)KD (M) GDF11 ActRIIB(20-134)-hFc 1.34e−6 1.13e−4 8.42e−11 ActRIIB(A24N20-134)-hFc 1.21e−6 6.35e−5 5.19e−11 ActRIIB(L79D 20-134)-hFc  6.7e−54.39e−4 6.55e−10 ActRIIB(L79E 20-134)-hFc  3.8e−5 2.74e−4 7.16e−10ActRIIB(R64K 20-134)-hFc 6.77e−5 2.41e−5 3.56e−11 GDF8ActRIIB(20-134)-hFc 3.69e−5 3.45e−5 9.35e−11 ActRIIB(A24N 20-134)-hFcActRIIB(L79D 20-134)-hFc 3.85e−5  8.3e−4 2.15e−9  ActRIIB(L79E20-134)-hFc 3.74e−5   9e−4 2.41e−9  ActRIIB(R64K 20-134)-hFc 2.25e−54.71e−5  2.1e−10 ActRIIB(R64K 20-129)-hFc 9.74e−4 2.09e−4 2.15e−9 ActRIIB(P129S, P130R 20- 1.08e−5  1.8e−4 1.67e−9  134)-hFc ActRIIB(K74A20-134)-hFc  2.8e−5 2.03e−5 7.18e−11 Activin A ActRIIB(20-134)-hFc5.94e6  1.59e−4 2.68e−11 ActRIIB(A24N 20-134)-hFc 3.34e6  3.46e−41.04e−10 ActRIIB(L79D 20-134)-hFc Low binding ActRIIB(L79E 20-134)-hFcLow binding ActRIIB(R64K 20-134)-hFc 6.82e6  3.25e−4 4.76e−11ActRIIB(R64K 20-129)-hFc 7.46e6  6.28e−4 8.41e−11 ActRIIB(P129S, P130R20- 5.02e6  4.17e−4 8.31e−11 134)-hFc

These data obtained in a cell-free assay confirm the cell-based assaydata, demonstrating that the A24N variant retains ligand-bindingactivity that is similar to that of the ActRIIB(20-134)-hFc molecule andthat the L79D or L79E molecule retains myostatin and GDF11 binding butshows markedly decreased (non-quantifiable) binding to activin A.

Other variants have been generated and tested, as reported inWO2006/012627 (incorporated herein by reference in its entirety). See,e.g., pp. 59-60, using ligands coupled to the device and flowingreceptor over the coupled ligands. Notably, K74Y, K74F, K74I (andpresumably other hydrophobic substitutions at K74, such as K74L), andD801, cause a decrease in the ratio of activin A (ActA) binding to GDF11binding, relative to the wild-type K74 molecule. A table of data withrespect to these variants is reproduced below:

Soluble ActRIIB-Fc variants binding to GDF11 and Activin A (Biacore ™assay) ActRIIB ActA GDF11 WT (64A) KD = 1.8e−7M KD = 2.6e−7M (+) (+) WT(64R) na KD = 8.6e−8M (+++) +15tail KD ~2.6e−8M KD = 1.9e−8M (+++)(++++) E37A * * R40A − − D54A − * K55A ++ * R56A * * K74A KD = 4.35e−9MKD = 5.3e−9M +++++ +++++ K74Y * −− K74F * −− K74I * −− W78A * * L79A + *D80K * * D80R * * D80A * * D80F * * D80G * * D80M * * D80N * * D80I * −−F82A ++ − * No observed binding −− <⅕ WT binding − ~½ WT binding + WT ++<2x increased binding +++ ~5x increased binding ++++ ~10x increasedbinding +++++ ~40x increased binding

Example 13: A GDF Trap Increases Red Blood Cell Levels In Vivo

Twelve-week-old male C57BL/6NTac mice were assigned to one of twotreatment groups (N=10). Mice were dosed with either vehicle or with avariant ActRIIB polypeptide (“GDF trap”) [ActRIIB(L79D 20-134)-hFc] bysubcutaneous injection (SC) at 10 mg/kg twice per week for 4 weeks. Atstudy termination, whole blood was collected by cardiac puncture intoEDTA containing tubes and analyzed for cell distribution using an HM2hematology analyzer (Abaxis, Inc).

Group Designation Dose Group N Mice Injection (mg/kg) Route Frequency 110 C57BL/6 PBS 0 SC Twice/week 2 10 C57BL/6 GDF trap 10 SC Twice/week[ActRIIB (L79D 20-134)-hFc]

Treatment with the GDF trap did not have a statistically significanteffect on the number of white blood cells (WBC) compared to the vehiclecontrols. Red blood cell (RBC) numbers were increased in the treatedgroup relative to the controls (see table below). Both the hemoglobincontent (HGB) and hematocrit (HCT) were also increased due to theadditional red blood cells. The average width of the red blood cells(RDWc) was higher in the treated animals, indicating an increase in thepool of immature red blood cells. Therefore, treatment with the GDF trapleads to increases in red blood cells, with no distinguishable effectson white blood cell populations.

Hematology Results RBC HGB HCT RDWc 10¹²/L (g/dL) (%) (%) PBS 10.7 ± 0.114.8 ± 0.6 44.8 ± 0.4 17.0 ± 0.1 GDF trap  12.4 ± 0.4**  17.0 ± 0.7* 48.8 ± 1.8*  18.4 ± 0.2** *= p < 0.05, **= p < 0.01

Example 14: A GDF Trap is Superior to ActRIIB-Fc for Increasing RedBlood Cell Levels in Vivo

Nineteen-week-old male C57BL/6NTac mice were randomly assigned to one ofthree groups. Mice were dosed with vehicle (10 mM Tris-buffered saline,TBS), wild-type ActRIIB(20-134)-mFc, or GDF trap ActRIIB(L79D20-134)-hFc by subcutaneous injection twice per week for three weeks.Blood was collected by cheek bleed at baseline and after three weeks ofdosing and analyzed for cell distribution using a hematology analyzer(HM2, Abaxis, Inc.)

Treatment with ActRIIB-Fc or the GDF trap did not have a significanteffect on white blood cell (WBC) numbers compared to vehicle controls.The red blood cell count (RBC), hematocrit (HCT), and hemoglobin levelswere all elevated in mice treated with GDF trap compared to either thecontrols or the wild-type construct (see table below). Therefore, in adirect comparison, the GDF trap promotes increases in red blood cells toa significantly greater extent than a wild-type ActRIIB-Fc protein. Infact, in this experiment, the wild-type ActRIIB-Fc protein did not causea statistically significant increase in red blood cells, suggesting thatlonger or higher dosing would be needed to reveal this effect.

Hematology Results after three weeks of dosing RBC HCT HGB (10¹²/ml) %g/dL TBS 11.06 ± 0.46 46.78 ± 1.9 15.7 ± 0.7 ActRIIB-mFc 11.64 ± 0.0949.03 ± 0.3 16.5 ± 1.5 GDF trap  13.19 ± 0.2**  53.04 ± 0.8**  18.4 ±0.3** **= p < 0.01

Example 15: Generation of a GDF Trap with Truncated ActRIIBExtracellular Domain

As described in Example 9, a GDF trap referred to as ActRIIB(L79D20-134)-hFc was generated by N-terminal fusion of TPA leader to theActRIIB extracellular domain (residues 20-134 in SEQ ID NO:1) containinga leucine-to-aspartate substitution (at residue 79 in SEQ ID NO:1) andC-terminal fusion of human Fc domain with minimal linker (three glycineresidues) (FIG. 16). A nucleotide sequence corresponding to this fusionprotein is shown in FIGS. 17A and 17B.

A GDF trap with truncated ActRIIB extracellular domain, referred to asActRIIB(L79D 25-131)-hFc, was generated by N-terminal fusion of TPAleader to truncated extracellular domain (residues 25-131 in SEQ IDNO:1) containing a leucine-to-aspartate substitution (at residue 79 inSEQ ID NO:1) and C-terminal fusion of human Fc domain with minimallinker (three glycine residues) (FIG. 18). A nucleotide sequencecorresponding to this fusion protein is shown in FIGS. 19A and 19B.

Example 16: Selective Ligand Binding by GDF Trap with Double-TruncatedActRIIB Extracelluar Domain

The affinity of GDF traps and other ActRIIB-hFc proteins for severalligands was evaluated in vitro with a Biacore™ instrument. Results aresummarized in the table below. Kd values were obtained by steady-stateaffinity fit due to the very rapid association and dissociation of thecomplex, which prevented accurate determination of k_(on), and k_(off).

Ligand Selectivity of ActRIIB-hFc Variants:

Activin A Activin B GDF11 Fusion Construct (Kd e−11) (Kd e−11) (Kd e−11)ActRIIB(L79 20-134)-hFc 1.6 1.2 3.6 ActRIIB(L79D 20-134)-hFc 1350.0 78.812.3 ActRIIB(L79 25-131)-hFc 1.8 1.2 3.1 ActRIIB(L79D 25-131)-hFc 2290.062.1 7.4

The GDF trap with a truncated extracellular domain, ActRIIB(L79D25-131)-hFc, equaled or surpassed the ligand selectivity displayed bythe longer variant, ActRIIB(L79D 20-134)-hFc, with pronounced loss ofactivin A binding, partial loss of activin B binding, and nearly fullretention of GDF11 binding compared to ActRIIB-hFc counterparts lackingthe L79D substitution. Note that truncation alone (without L79Dsubstitution) did not alter selectivity among the ligands displayed here[compare ActRIIB(L79 25-131)-hFc with ActRIIB(L79 20-134)-hFc].

Example 17: Generation of ActRIIB(L79D 25-131)-hFc with AlternativeNucleotide Sequences

To generate ActRIIB(L79D 25-131)-hFc, the human ActRIIB extracellulardomain with an aspartate substitution at native position 79 (SEQ IDNO:1) and with N-terminal and C-terminal truncations (residues 25-131 inSEQ ID NO: 1) was fused N-terminally with a TPA leader sequence insteadof the native ActRIIB leader and C-terminally with a human Fc domain viaa minimal linker (three glycine residues) (FIG. 18). One nucleotidesequence encoding this fusion protein is shown in FIGS. 19A and 19B (SEQID NO: 42), and an alternative nucleotide sequence encoding exactly thesame fusion protein is shown in FIGS. 22A and 22B (SEQ ID NO: 46). Thisprotein was expressed and purified using the methodology described inExample 9.

Example 18: GDF Trap with a Truncated ActRIIB Extracellular DomainIncreases Proliferation of Erythroid Progenitors in Mice

ActRIIB(L79D 25-131)-hFc was evaluated to determine its effect onproliferation of erythroid progenitors. Male C57BL/6 mice (8 weeks old)were treated with ActRIIB(L79D 25-131)-hFc (10 mg/kg, s.c.; n=6) orvehicle (TBS; n=6) on Days 1 and 4, then euthanized on Day 8 forcollection of spleens, tibias, femurs, and blood. Cells of the spleenand bone marrow were isolated, diluted in Iscove's modified Dulbecco'smedium containing 5% fetal bovine serum, suspended in specializedmethylcellulose-based medium, and cultured for either 2 or 12 days toassess levels of clonogenic progenitors at the colony-formingunit-erythroid (CFU-E) and burst forming unit-erythroid (BFU-E) stages,respectively. Methylcellulose-based medium for BFU-E determination(MethoCult M3434, Stem Cell Technologies) included recombinant murinestem cell factor, interleukin-3, and interleukin-6, which were notpresent in methylcellulose medium for CFU-E determination (MethoCultM3334, Stem Cell Technologies), while both media containederythropoietin, among other constituents. For both BFU-E and CFU-E, thenumber of colonies were determined in duplicate culture plates derivedfrom each tissue sample, and statistical analysis of the results wasbased on the number of mice per treatment group.

Spleen-derived cultures from mice treated with ActRIIB(L79D 25-131)-hFchad twice the number of CFU-E colonies as did corresponding culturesfrom control mice (P<0.05), whereas the number of BFU-E colonies did notdiffer significantly with treatment in vivo. The number of CFU-E orBFU-E colonies from bone marrow cultures also did not differsignificantly with treatment. As expected, increased numbers of CFU-Ecolonies in spleen-derived cultures were accompanied by highlysignificant (P<0.001) changes in red blood cell level (11.6% increase),hemoglobin concentration (12% increase), and hematocrit level (11.6%increase) at euthanasia in mice treated with ActRIIB(L79D 25-131)-hFccompared to controls. These results indicate that in vivo administrationof a GDF trap with truncated ActRIIB extracellular domain can stimulateproliferation of erythroid progenitors as part of its overall effect toincrease red blood cell levels.

GDF trap fusion proteins have been further demonstrated to be effectivein increasing red blood cell levels in various models of anemiaincluding, for example, chemotherapy-induced anemia, nephrectomy-inducedanemia, and in a blood loss anemia (see, e.g., International PatentApplication Publication No. WO 2010/019261).

Example 19: GDF Trap with Truncated ActRIIB Extracellular DomainIncreases Levels of Red Blood Cells in Non-Human Primates

Two GDF Traps, ActRIIB(L79D 20-134)-hFc and ActRIIB(L79D 25-131)-hFc,were evaluated for their ability to stimulate red blood cell productionin cynomolgus monkeys. Monkeys were treated subcutaneously with GDF trap(10 mg/kg; n=4 males/4 females), or vehicle (n=2 males/2 females) onDays 1 and 8. Blood samples were collected on Days 1 (pretreatmentbaseline), 3, 8, 15, 29, and 44, and were analyzed for red blood celllevels (FIG. 24), hematocrit (FIG. 25), hemoglobin levels (FIG. 26), andreticulocyte levels (FIG. 27). Vehicle-treated monkeys exhibiteddecreased levels of red blood cells, hematocrit, and hemoglobin at allpost-treatment time points, an expected effect of repeated bloodsampling. In contrast, treatment with ActRIIB(L79D 20-134)-hFc orActRIIB(L79D 25-131)-hFc increased these parameters by the firstpost-treatment time point (Day 3) and maintained them at substantiallyelevated levels for the duration of the study (FIGS. 24-26).Importantly, reticulocyte levels in monkeys treated with ActRIIB(L79D20-134)-hFc or ActRIIB(L79D 25-131)-hFc were substantially increased atDays 8, 15, and 29 compared to vehicle (FIG. 27). This resultdemonstrates that GDF trap treatment increased production of red bloodcell precursors, resulting in elevated red blood cell levels.

Taken together, these data demonstrate that truncated GDF traps, as wellas a full-length variants, can be used as selective antagonists of GDF11and potentially related ligands to increase red blood cell formation invivo.

Example 20: GDF Trap Derived from ActRIIB5

Others have reported an alternate, soluble form of ActRIIB (designatedActRIIB5), in which exon 4, including the ActRIIB transmembrane domain,has been replaced by a different C-terminal sequence (see, e.g., WO2007/053775).

The sequence of native human ActRIIB5 without its leader is as follows:

(SEQ ID NO: 49) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEGPWASTTIPSGGPEATAAAGDQGSGALWLCLEGPAHE

An leucine-to-aspartate substitution, or other acidic substitutions, maybe performed at native position 79 (underlined) as described toconstruct the variant ActRIIB5(L79D), which has the following sequence:

(SEQ ID NO: 50) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEGPWASTTIPSGGPEATAAAGDQGSGALWLCLEGPAHE

This variant may be connected to human Fc (double underline) with a TGGGlinker (single underline) to generate a human ActRIIB5(L79D)-hFc fusionprotein with the following sequence:

(SEQ ID NO: 51) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWDDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEGPWASTTIPSGGPEATAAAGDQGSGALWLCLEGPAHETGGG THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK.

This construct may be expressed in CHO cells.

Example 21: Effects in Mice of Combined Treatment with EPO and a GDFTrap with a Truncated ActRIIB Extracellular Domain

EPO induces formation of red blood cells by increasing the proliferationof erythroid precursors, whereas GDF traps could potentially affectformation of red blood cells in ways that complement or enhance EPO'seffects. Therefore, Applicants investigated the effect of combinedtreatment with EPO and ActRIIB(L79D 25-131)-hFc on erythropoieticparameters. Male C57BL/6 mice (9 weeks old) were given a single i.p.injection of recombinant human EPO alone (epoetin alfa, 1800 units/kg),ActRIIB(L79D 25-131)-hFc alone (10 mg/kg), both EPO and ActRIIB(L79D25-131)-hFc, or vehicle (Tris-buffered saline). Mice were euthanized 72h after dosing for collection of blood, spleens, and femurs.

Spleens and femurs were processed to obtain erythroid precursor cellsfor flow cytometric analysis. After removal, the spleen was minced inIscove's modified Dulbecco's medium containing 5% fetal bovine serum andmechanically dissociated by pushing through a 70-μm cell strainer withthe plunger from a sterile 1-mL syringe. Femurs were cleaned of anyresidual muscle or connective tissue and ends were trimmed to permitcollection of marrow by flushing the remaining shaft with Iscove'smodified Dulbecco's medium containing 5% fetal bovine serum through a21-gauge needle connected to a 3-mL syringe. Cell suspensions werecentrifuged (2000 rpm for 10 min) and the cell pellets resuspended inPBS containing 5% fetal bovine serum. Cells (10⁶) from each tissue wereincubated with anti-mouse IgG to block nonspecific binding, thenincubated with fluorescently labeled antibodies against mousecell-surface markers CD71 (transferrin receptor) and Ter119 (an antigenassociated with cell-surface glycophorin A), washed, and analyzed byflow cytrometry. Dead cells in the samples were excluded from analysisby counterstaining with propidium iodide. Erythroid differentiation inspleen or bone marrow was assessed by the degree of CD71 labeling, whichdecreases over the course of differentiation, and Ter119 labeling, whichis increased during terminal erythroid differentiation beginning withthe proerythroblast stage (Socolovsky et al., 2001, Blood 98:3261-3273;Ying et al., 2006, Blood 108:123-133). Thus, flow cytometry was used todetermine the number of proerythroblasts (CD71^(high)Ter119^(low)),basophilic erythroblasts (CD71^(high)Ter119^(high)),polychromatophilic+orthochromatophilic erythroblasts(CD71^(med)Ter119^(high)), and late orthochromatophilicerythroblasts+reticulocytes (CD71^(low)Ter119^(high)), as described.

Combined treatment with EPO and ActRIIB(L79D 25-131)-hFc led to asurprisingly vigorous increase in red blood cells. In the 72-h timeframe of this experiment, neither EPO nor ActRIIB(L79D 25-131)-hFc aloneincreased hematocrit significantly compared to vehicle, whereas combinedtreatment with the two agents led to a nearly 25% increase in hematocritthat was unexpectedly synergistic, i.e., greater than the sum of theirseparate effects (FIG. 28). Synergy of this type is generally consideredevidence that individual agents are acting through different cellularmechanisms. Similar results were also observed for hemoglobinconcentrations (FIG. 29) and red blood cell concentrations (FIG. 30),each of which was also increased synergistically by combined treatment.

Analysis of erythroid precursor levels revealed a more complex pattern.In the mouse, the spleen is considered the primary organ responsible forinducible (“stress”) erythropoiesis. Flow cytometric analysis of splenictissue at 72 h revealed that EPO markedly altered the erythropoieticprecursor profile compared to vehicle, increasing the number ofbasophilic erythroblasts by more than 170% at the expense of lateprecursors (late orthochromatophilic erythroblasts+reticulocytes), whichdecreased by more than one third (FIG. 31). Importantly, combinedtreatment increased basophilic erythroblasts significantly compared tovehicle, but to a lesser extent than EPO alone, while supportingundiminished maturation of late-stage precursors (FIG. 31). Thus,combined treatment with EPO and ActRIIB(L79D 25-131)-hFc increasederythropoiesis through a balanced enhancement of precursor proliferationand maturation. In contrast to spleen, the precursor cell profile inbone marrow after combined treatment did not differ appreciably fromthat after EPO alone. Applicants predict from the splenic precursorprofile that combined treatment would lead to increased reticulocytelevels and would be accompanied by sustained elevation of mature redblood cell levels if the experiment were extended beyond 72 h.

Taken together, these findings demonstrate that a GDF trap with atruncated ActRIIB extracellular domain can be administered incombination with EPO to synergistically increase red blood cellformation in vivo. Acting through a complementary but undefinedmechanism, a GDF trap can moderate the strong proliferative effect of anEPO receptor activator alone and still permit target levels of red bloodcells to be attained with lower doses of an EPO receptor activator,thereby avoiding potential adverse effects or other problems associatedwith higher levels of EPO receptor activation.

Example 22: Effect of a GDF Trap on Ineffective Erythropoiesis andAnemia in a Mouse Model of MDS

Applicants investigated effects of the GDF trap ActRIIB(L79D 25-131)-mFc(RAP-536) in the NUP98-HOXD13 mouse model of MDS, which is characterizedby abortive precursor maturation and ineffective hematopoiesis. In thismodel, disease severity increases with age, eventually progressing toacute myeloid leukemia in the majority of mice, and they have a meanlife span of approximately 14 months. Starting at approximately 4 monthsof age, these mice exhibit anemia, leukopenia, ineffectiveerythropoiesis, and bone marrow that is normocellular to hypercellular[Lin et al. (2005) Blood 106:287-295]. To monitor the effects of chronicadministration, MDS mice were treated with RAP-536 (10 mg/kg, s.c.) orvehicle twice weekly beginning at 4 months of age and continuing for 7months, while blood samples (50 μL) were collected at baseline andmonthly thereafter for complete blood count analysis. As expected,6-month-old MDS mice developed severe anemia compared to wild-type mice(FIG. 32A), and bone marrow analyses revealed increased numbers oferythroid precursors (FIG. 32A) and a lower myeloid/erythroid (M/E)ratio [Suragani et al. (2014) Nat Med 20:408-414] in MDS mice comparedto age-matched FVB wild-type mice, indicative of ineffectiveerythropoiesis. In 6-month-old MDS mice, treatment with RAP-536significantly increased RBC count (by 16.9%) and hemoglobinconcentration (by 12.5%) (FIG. 32A), reduced erythroid precursor cellcount in bone marrow (FIG. 32A), and normalized the M/E ratio to that ofwild-type mice [Suragani et al. (2014) Nat Med 20:408-414].

In MDS mice at 12 months of age, RAP-536 treatment significantlyincreased RBC count (by 18.3%) and hemoglobin level (by 13.0%) (FIG.32B), reduced erythroid precursor cell count (FIG. 32B), and improvedthe M:E ratio [Suragani et al. (2014) Nat Med 20:408-414], as comparedto vehicle. RAP-536 treatment did not affect the absolute number ofmyeloid precursors. Flow cytometry confirmed that RAP-536 treatmentreduced erythroid hyperplasia in MDS mice at both ages. A time-courseanalysis in MDS mice treated with RAP-536 for 7 months showed asustained elevation in RBC numbers for the duration of the study[Suragani et al. (2014) Nat Med 20:408-414]. Together, these resultsindicate that treatment with a GDF trap ameliorates anemia, erythroidhyperplasia and ineffective erythropoiesis in MDS mice regardless ofdisease severity.

Example 23: Cytologic and Genetic Signatures in MDS PatientsTherapeutically Responsive to a GDF Trap

A recombinant fusion protein containing modified activin receptor typeIIB and IgG Fc [ActRIIB(L79D 25-131)-hFc, also known as luspatercept orACE-536] is being developed for the treatment of anemias due toineffective erythropoiesis such as myelodysplastic syndromes (MDS).Patients with MDS often have elevated levels of EPO and may benon-responsive or refractory to erythropoiesis-stimulating agents(ESAs). MDS patients have also been shown to have increased serum levelsof GDF11 [Suragani et al. (2014) Nat Med 20:408-414] and increased Smad2/3 signaling in the bone marrow [Zhou et al. (2008) Blood112:3434-3443]. ActRIIB(L79D 25-131)-hFc binds to ligands in the TGFβsuperfamily, including GDF11, inhibits Smad2/3 signaling, and promoteslate-stage erythroid differentiation via a mechanism distinct from ESAs.A murine version, ActRIIB(L79D 25-131)-mFc, reduced Smad2 signaling,increased hemoglobin (Hb) levels, and decreased bone marrow erythroidhyperplasia in a mouse model of MDS [Suragani et al. (2014) Nat Med20:408-414]. In a healthy-volunteer study, ActRIIB(L79D 25-131)-hFc waswell-tolerated and increased Hb levels [Attie et al. (2014) Am J Hematol89:766-770].

Applicants are conducting an ongoing, phase 2, multicenter, open-label,dose-finding study to evaluate the effects of ActRIIB(L79D 25-131)-hFcon anemia in patients with Low or Int-1 risk MDS who have either hightransfusion burden (HTB, defined as ≥4 units RBC per 8 weeks prior tobaseline) or low transfusion burden (LTB, defined as <4 units RBC per 8weeks prior to baseline). Study outcomes include erythroid response(either Hb increase in LTB patients or reduced transfusion burden in HTBpatients), safety, tolerability, pharmacokinetics, and pharmacodynamicbiomarkers. Inclusion criteria include: Low or Int-1 risk MDS, age≥18yr, anemia (defined as either being HTB patient or having baselineHb<10.0 g/dL in LTB patient), EPO>500 U/L or nonresponsive/refractory toESAs, no prior azacitidine or decitabine, and no current treatment withESA, granulocyte colony-stimulating factor (G-CSF),granulocyte-macrophage colony-stimulating factor (GM-CSF), orlenalidomide, thalidomide or pomalidomide. In the dose-escalation phase,ActRIIB(L79D 25-131)-hFc was administered by subcutaneous injection onceevery 3 weeks in seven sequential cohorts (n=3-6) at dose levels of0.125, 0.25, 0.5, 0.75, 1.0, 1.33 and 1.75 mg/kg for up to 5 doses witha 3-month follow-up.

Data were available for 26 patients (seven LTB/19 HTB). Median age was71 yr (range: 27-88 yr), 50% were female, 54% had prior EPO therapy, and15% had prior lenalidomide. Patient classification by WHO subtype was asfollows: 15% RARS, 46% RCMD-RS, 15% RCMD, 15% RAEB-1 (including twopatients with ≥15% ring sideroblasts) and 8% del (5q). Mean (SD)baseline Hgb for the LTB patients (n=7) was 9.1 (0.4) g/dL. Mean (SD)units RBC transfused in the 8 weeks prior to treatment was 0.9 (1.1)units for the LTB patients and 6.3 (2.4) units for the HTB patients. Twoof the seven LTB patients had an increase in mean Hb≥1.5 g/dL over 8weeks compared to baseline. Mean maximum Hb increase in the LTB patientswas 0.8, 1.0, 2.2, and 3.5 g/dL in the 0.125 (n=1), 0.25 (n=1), 0.75(n=3), and 1.75 (n=2) mg/kg dose groups, respectively. Six of the sevenLTB patients achieved RBC transfusion independence (RBC-TI) for ≥8 weeksduring the study. The dose levels ranging from 0.75 mg/kg to 1.75 mg/kgwere deemed to be active doses. Among the five patients in the activedose groups, four (80%) achieved the pre-specified endpoint of Hgbincrease of ≥1.5 g/dl for ≥2 weeks. Two patients (40%) achieved a HI-Eresponse [International Working Group; Cheson et al. (2000) Blood96:3671-3674; Cheson et al. (2006) Blood 108:419-425], defined as an Hgbincrease of ≥1.5 g/dl for ≥8 weeks in LTB patients. In HTB patients, theHI-E response is defined as a reduction in transfusion burden of atleast four units of red blood cells transfused over an 8 week period ascompared to the 8 weeks prior to study start. In the active dose groups,five of 12 (42%) HTB patients met the pre-specified endpoint of areduction of ≥4 RBC units or ≥50% reduction in RBC units transfused overan 8-week interval during the treatment period compared to the 8 weeksprior to treatment, and the same patients (five of 12, 42%) achieved aHI-E response; three of 12 (25%) of HTB patients in the active dosegroups achieved RBC-TI≥8 weeks during the study. Increases in neutrophilcount following study drug administration were observed in somepatients. Finally, ActRIIB(L79D 25-131)-hFc was generally welltolerated. No related serious adverse events have been reported to date.The most frequent adverse events regardless of causality were: diarrhea(n=4, grade 1/2), bone pain, fatigue, muscle spasms, myalgia, andnasopharyngitis (n=3 each, grade 1/2).

Assessment of bone marrow aspirates demonstrated an association betweenthe presence of ring sideroblasts (considered positive if ≥15% oferythroid precursors exhibited ring sideroblast morphology) andresponsiveness to ActRIIB(L79D 25-131)-hFc in the active dose groups(0.75-1.75 mg/kg). The overall response rate (using HI-E criteria,described above) across both LTB and HTB patients was seven of 17 (41%).Among patients positive for ring sideroblasts at baseline, seven of 13(54%) patients achieved a HI-E response, and notably none of the fourpatients negative for ring sideroblasts at baseline achieved a HI-Eresponse.

Bone marrow aspirates from patients were also evaluated for the presenceof mutations in 21 different genes that are known to harbor mutations(primarily somatic mutations) associated with MDS. Genomic DNA wasisolated from bone marrow aspirates, selected coding regions of the 21genes were amplified by polymerase chain reaction, and the DNA sequencesof these regions were determined using next-generation sequencing(Myeloid Molecular Profile 21-gene panel, Genoptix, Inc., Carlsbad,Calif.). This analysis examined activated signaling genes (KIT, JAK2,NRAS, CBL, and MPL), transcription factors (RUNX1, ETV6), epigeneticgenes (IDH1, IDH2, TET2, DNMT3A, EZH2, ASXL1, and SETBP1), RNA splicinggenes (SF3B1, U2AF1, ZRSR2, and SRSF2), and tumor suppressors/others(TP53, NPM1, PHF6). Analysis of SF3B1 specifically targeted exons 13-16.Of these 21 MDS-associated genes evaluated, mutations in SF3B1 were morefrequently detected in bone marrow cells in the responsive patients thanin the nonresponsive patients. Individual SF3B1 mutations detected inthese patients are shown in the following table. The same mutationsometimes occurred in multiple patients.

Nucleotide Amino Acid Nucleotide Substitution Substitution Exon 1873 C →T R625C 14 1874 G → T R625L 14 1986 C → G H662Q 14 2098 A → G K700E 152342 A → G D781G 16

In patients with SF3B1 mutations in the active dose groups, six of nine(67%) achieved HI-E responses, including all three patients thatachieved transfusion independence for greater than 8 weeks. In patientsnot having an SF3B1 mutation, only one of eight (13%) achieved a HI-Eresponse. Mutations in SF3B1 are frequently observed in MDS patientswith ring sideroblasts and are associated with ineffectiveerythropoiesis.

The initial analysis of the clinical trial in process, presented above,was confirmed in a later analysis, for which data were available for 44patients (15 LTB/29 HTB). Median age was 71 yr (range: 27-88 yr), 43%were female, 61% had prior EPO therapy, and 21% had prior lenalidomide.Mean baseline Hgb for the LTB patients (n=15) was 9.0 (range: 6.8-10.1)g/dL. Mean units RBC transfused in the 8 weeks prior to treatment was 2(range 2-2) units for the 6 LTB patients that received transfusions and6 (range: 4-14) units for the HTB patients. The dose levels ranging from0.75 mg/kg to 1.75 mg/kg were deemed to be active doses. Among the 35LTB and HTB patients in the active dose groups, 22 (63%) achieved thepre-specified endpoint of Hgb increase of ≥1.5 g/dl for ≥2 weeks for LTBpatients and ≥4 unit or 50% reduction in transfusions over 8 weeks forHTB patients. 19 of 35 patients (54%) in the active dose groups achieveda HI-E response [International Working Group; Cheson et al. (2000) Blood96:3671-3674; Cheson et al. (2006) Blood 108:419-425], defined as an Hgbincrease of ≥1.5 g/dl for ≥8 weeks in LTB patients and defined as areduction of ≥4 RBC units or ≥50% reduction in RBC units transfused overan 8-week interval during the treatment period compared to the 8 weeksprior to treatment in HTB patients. 10/28 (36%) patients in the activedose groups that had baseline transfusions achieved transfusionindependence for a period of at least 8 weeks. Assessment of bone marrowaspirates demonstrated an association between the presence of ringsideroblasts (considered positive if ≥15% of erythroid precursorsexhibited ring sideroblast morphology) or a mutation in the SF3B1 geneand responsiveness to ActRIIB(L79D 25-131)-hFc in the active dose groups(0.75-1.75 mg/kg). The overall response rate (using HI-E criteria,described above) across both LTB and HTB patients was 19/35 (54%). Amongpatients positive for ring sideroblasts at baseline, 19/30 (63%)patients achieved a HI-E response, and notably none of the four patientsnegative for ring sideroblasts at baseline achieved a HI-E response.Among patients positive for SF3B1 mutation at baseline, 16/22 (73%)patients achieved a HI-E response, and notably only 3/13 (23%) patientsnegative for SF3B1 mutation at baseline achieved a HI-E response.Finally, ActRIIB(L79D 25-131)-hFc was generally well tolerated. The mostfrequent adverse events regardless of causality were: diarrhea,nasopharyngitis, myalgia, bone pain, bronchitis, headache and and musclespasms. Two possibly related serious adverse events (SAEs) werereported: grade 3 muscle pain; grade 3 worsening of general condition.One possibly related non-serious grade 3 adverse event of blast cellcount increase was reported.

A further data assessment conducted at a later date extended andgenerally confirmed the above results. Overall, 24 of 49 patients (49%)in the active dose groups achieved an HI-E response, defined in patientswith low-transfusion burden (LTB) as an increase in hemoglobinconcentration of ≥1.5 g/dL for ≥8 weeks and defined in patients withhigh-transfusion burden (HTB) as a reduction of ≥4 RBC units, or areduction of ≥50% RBC units, transfused over an 8-week interval duringthe treatment period compared to the 8 weeks prior to treatment.Fourteen of 40 patients (35%) in the active dose groups that hadbaseline transfusions achieved transfusion independence for a period ofat least 8 weeks.

An assessment of response rate in the presence of certain somatic genemutations was also conducted for patients in the active dose groups.Mutations in SF3B1 were detected more frequently in bone marrow cellsfrom the responsive patients than from the nonresponsive patients.Eighteen of 30 patients (60%) in the active dose groups with an SF3B1mutation achieved an HI-E response, whereas only 6 of 19 such patients(32%) without a mutation detected in this gene achieved an HI-Eresponse. Individual SF3B1 mutations detected in these patients areshown in the following table. The same mutation sometimes occurred inmultiple patients.

Nucleotide Nucleotide Change AA Change Exon 1868 A → G Y623C 14 1873 C →T R625C 14 1874 G → T R625L 14 1986 C → G H662Q 14 2098 A → G K700E 152342 A → G D781G 16 2347 G → A E783K 16

Similarly, mutations in DNMT3A were detected more frequently in bonemarrow cells from responsive patients in the active dose groups thanfrom the nonresponsive patients. Seven of 11 patients (64%) in theactive dose groups with a DNMT3A mutation achieved an HI-E response,whereas 17 of 38 such patients (45%) without a mutation detected in thisgene achieved an HI-E response. Individual DNMT3A mutations detected inthese patients are shown in the following table (IVS refers to intronicmutations and X indicates formation of a premature stop codon).

Nucleotide Nucleotide Change AA Change Exon 1308 C → A Y436X 10 IVS 2082+2 T → C — — 2193_2195 del CTT In frame 18 2216 del A Frame shift 18 IVS2322 +2 T → C — — 2644 C → T R882C 22 2645 G → A R882H 22 2678 G → AW893X 22 2711 C → T P904L 22 2714 T → C L905P 22

Similarly, mutations in TET2 were detected more frequently in bonemarrow cells from responsive patients in the active dose groups thanfrom the nonresponsive patients. Eleven of 20 patients (55%) in theactive dose groups with a TET2 mutation achieved an HI-E response,whereas 13 of 29 such patients (45%) without a mutation detected in thisgene achieved an HI-E response. Individual TET2 mutations detected inthese patients (excluding known polymorphisms) are shown in thefollowing table.

Nucleotide Nucleotide Change AA Change Exon   73 del T Frame shift 1 139 G → C E47Q 1  735 del C Frame shift 1 1201_1202 ins ACCACCACCACFrame shift 1 1337 del T Frame shift 1 1588_1591 del CAGC Frame shift 11648 C → T R550X 1 1842_1843 ins G Frame shift 1 2145 del C Frame shift1 2305 del C Frame shift 1 2784 del T Frame shift 1 3025 C → T Q1009X 13727_3729 del AAA In frame 4 3731_3738 del TCTACTCG Frame shift 4 3821 A→ G Q1274R 5 3854_3856 del TCT In frame 5 3871 T → A W1291R 5 IVS 3955−2 A → G — — 4011 T → A Y1337X 6 4108 G → A G1370R 7 4109 G → A G1370E 74160 A → G N1387S 7 4209 del T Frame shift 8 4210 C → T R1404X 84211_4217 del GAGAATT Frame shift 8 4546 C → T R1516X 9 4954 C → TQ1652X 9 5168 del C Frame shift 9 5170 T → C Y1724H 9 5576_5582 delTTGGGGG Frame shift 9

Mutations in other genes were detected in bone marrow cells fromresponsive patients with a frequency similar to that in cells fromnonresponsive patients. For example, 4 of 8 patients (50%) in the activedose groups with an ASXL1 mutation achieved an HI-E response, while asimilar percentage of such patients (20 of 41, 49%) without a mutationdetected in this gene achieved an HI-E response.

These results indicate that patients with MDS exhibiting≥15% ringsideroblasts (and patients with other forms of sideroblastic anemia)and/or at least one mutation in SF3B1 are more likely to respondtherapeutically to ActRIIB(L79D 25-131)-hFc than MDS patients with <15%ring sideroblasts and/or no mutation in SF3B1. Similarly, patientsexhibiting exhibiting≥15% ring sideroblasts (and patients with otherforms of sideroblastic anemia) and/or at least one mutation in DNMT3A orTET2 are more likely to respond therapeutically to ActRIIB(L79D25-131)-hFc than MDS patients with <15% ring sideroblasts and/or nomutation in DNMT3A or TET2. Based on these data, selective treatment ofany of these patient subgroups is expected to greatly increase thebenefit/risk ratio of treatment with ActRII inhibitors.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A method for treating sideroblastic anemia in a human patient,comprising administering to a patient in need thereof a polypeptidecomprising an amino acid sequence that is at least 90% identical toamino acids 29-109 of SEQ ID NO: 1, wherein the polypeptide comprises anacidic amino acid at the amino acid position corresponding to position79 of SEQ ID NO: 1, and wherein the patient has bone marrow cells thattest positive for one or more mutations in one or more of SF3B1, DNMT3Aor TET2. 2-11. (canceled)
 12. The method of claim 1, wherein the patienthas undesirably high levels of endogenous EPO.
 13. The method of claim1, wherein the patient has previously been treated with one or more EPOreceptor agonists.
 14. The method of claim 13, wherein the patient hasan inadequate response to the EPO receptor agonist.
 15. The method ofclaim 13, wherein the patient is no longer responsive to the EPOreceptor agonist.
 16. The method of claim 13, wherein the EPO receptoragonist is EPO.
 17. The method of claim 1, wherein the treatmentincreases red blood cell levels.
 18. The method of claim 1, wherein thetreatment increases hemoglobin levels.
 19. The method of claim 18,wherein the treatment results in an increase in hemoglobin of ≥1.5 g/dLfor ≥two weeks.
 20. The method of claim 18, wherein the treatmentresults in an increase in hemoglobin of ≥1.5 g/dL for ≥eight weeks. 21.The method of claim 1, wherein the patient has been administered one ormore blood cell transfusions prior to the start of treatment.
 22. Themethod of claim 1, wherein the patient is a low transfusion burdenpatient.
 23. The method of claim 1, wherein the patient is a hightransfusion burden patient.
 24. The method of claim 1, wherein thetreatment decreases blood cell transfusion burden.
 25. The method ofclaim 24, wherein the treatment decreases blood cell transfusion by ≥50%for at least four weeks relative to the equal time prior to start oftreatment.
 26. The method of claim 24, wherein the treatment decreasesblood cell transfusion by ≥50% for at least eight weeks relative to theequal time prior to start of treatment.
 27. The method of claim 1,wherein the patient has myelodysplastic syndrome.
 28. The method ofclaim 27, wherein the patient has an International Prognostic ScoringSystem (IPSS) or IPSS-R score of low or intermediate.
 29. The method ofclaim 1, wherein the sideroblastic anemia patient has at least 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%ring blasts as a percentage of bone marrow erythroid precursors in hisor her bone marrow.
 30. The method of claim 1, wherein the treatmentincreases neutrophil levels.
 31. The method of claim 1, wherein thepatient has bone marrow cells that test positive for one or moremutations in SF3B1.
 32. The method of claim 1, wherein the patient hasbone marrow cells that test positive for one or more mutations inDNMT3A.
 33. The method of claim 1, wherein the patient has bone marrowcells that test positive for one or more mutations in TET2.
 34. Themethod of claim 1, wherein the treatment decreases iron overload.35-195. (canceled)
 196. The method of claim 1, wherein the polypeptidecomprises an amino acid sequence that is at least 95% identical to aminoacids 29-109 of SEQ ID NO:
 1. 197. The method of claim 1, wherein thepolypeptide comprises amino acids 29-109 of SEQ ID NO: 1, but whereinthe polypeptide comprises an acidic amino acid at the amino acidposition corresponding to position 79 of SEQ ID NO:
 1. 198. The methodof claim 1, wherein the polypeptide comprises an amino acid sequencethat is at least 90% identical to amino acids 25-131 of SEQ ID NO: 1.199. The method of claim 1, wherein the polypeptide comprises an aminoacid sequence that is at least 95% identical to amino acids 25-131 ofSEQ ID NO:
 1. 200. The method of claim 1, wherein the polypeptidecomprises amino acids 25-131 of SEQ ID NO: 1, but wherein thepolypeptide comprises an acidic amino acid at the amino acid positioncorresponding to position 79 of SEQ ID NO:
 1. 201. The method of claim1, wherein the polypeptide comprises an amino acid sequence that is atleast 90% identical to the sequence of SEQ ID NO:
 44. 202. The method ofclaim 1, wherein the polypeptide comprises an amino acid sequence thatis at least 95% identical to the sequence of SEQ ID NO:
 44. 203. Themethod of claim 1, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:
 44. 204. The method of claim 1, wherein theacidic amino acid is an aspartic acid.
 205. The method of claim 1,wherein the acidic amino acid is a glutamic acid.